Endothelial cell expression patterns

ABSTRACT

To gain a better understanding of tumor angiogenesis, new techniques for isolating endothelial cells (ECs) and evaluating gene expression patterns were developed. When transcripts from ECs derived from normal and malignant colorectal tissues were compared with transcripts from non-endothelial cells, over 170 genes predominantly expressed in the endothelium were identified. Comparison between normal- and tumor-derived endothelium revealed 79 differentially expressed genes, including 46 that were specifically elevated in tumor-associated endothelium. Experiments with representative genes from this group demonstrated that most were similarly expressed in the endothelium of primary lung, breast, brain, and pancreatic cancers as well as in metastatic lesions of the liver. These results demonstrate that neoplastic and normal endothelium in humans are distinct at the molecular level, and have significant implications for the development of anti-angiogenic therapies in the future.

This application is a continuation of U.S. Ser. No. 09/918,715, filedAug. 1, 2001. This application claims the benefit of provisionalapplications Ser. Nos. 60/222,599 filed Aug. 2, 2000, 60/224,360 filedAug. 11, 2000, and 60/282,850 filed Apr. 11, 2001, the disclosures ofwhich are expressly incorporated herein.

The U.S. government retains certain rights in the invention by virtue ofthe provisions of National Institutes of Heath grants CA57345 andCA43460, which supported this work.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of angiogenesis andanti-angiogenesis. In particular, it relates to genes which arecharacteristically expressed in tumor endothelial and normal endothelialcells.

BACKGROUND OF THE INVENTION

It is now widely recognized that tumors require a blood supply forexpansive growth. This recognition has stimulated a profusion ofresearch on tumor angiogenesis, based on the idea that the vasculaturein tumors represents a potential therapeutic target. However, severalbasic questions about tumor endothelium remain unanswered. For example,are vessels of tumors qualitatively different from normal vessels of thesame tissue? What is the relationship of tumor endothelium toendothelium of healing wounds or other physiological or pathologicalforms of angiogenesis? The answers to these questions critically impacton the potential for new therapeutic approaches to inhibit angiogenesisin a specific manner.

There is a continuing need in the art to characterize the vasculature oftumors relative to normal vasculature so that any differences can beexploited for therapeutic and diagnostic benefits.

One technique which can be used to characterize gene expression, or moreprecisely gene transcription, is termed serial analysis of geneexpression (SAGE). Briefly, the SAGE approach is a method for the rapidquantitative and qualitative analysis of mRNA transcripts based upon theisolation and analysis of short defined sequence tags (SAGE Tags)corresponding to expressed genes. Each Tag is a short nucleotidesequences (9-17 base pairs in length) from a defined position in thetranscript. In the SAGE method, the Tags are dimerized to reduce biasinherent in cloning or amplification reactions. (See, U.S. Pat. No.5,695,937) SAGE is particularly suited to the characterization of genesassociated with vasculature stimulation or inhibition because it iscapable of detecting rare sequences, evaluating large numbers ofsequences at one time, and to provide a basis for the identification ofpreviously unknown genes.

SUMMARY OF THE INVENTION

One embodiment of the invention provides an isolated molecule comprisingan antibody variable region which specifically binds to an extracellulardomain of a TEM protein selected from the group consisting of: 1, 3, 9,17, 19, and 44, as shown in SEQ ID NO: 196, 200, 212, 230, 232, and 271,respectively. The molecule can be, for example, an in tact antibodymolecule, a single chain variable region (ScFv), a monoclonal antibody,a humanized antibody, or a human antibody. The molecule can optionallybe bound to a cytotoxic moiety, bound to a therapeutic moiety, bound toa detectable moiety, or bound to an anti-tumor agent.

According to another embodiment of the invention a method of inhibitingneoangiogenesis is provided. An effective amount of an isolated moleculecomprising an antibody variable region which specifically binds to anextracellular domain of a TEM protein selected from the group consistingof: 1, 3, 9, 17, 19, 22, and 44, as shown in SEQ ID NO: 196, 200, 212,230, 232, 238, and 271, respectively, is administered to a subject inneed thereof. Neoangiogenesis is consequently inhibited. The subject maybear a vascularized tumor, may have polycystic kidney disease, may havediabetic retinopathy, may have rheumatoid arthritis, may have psoriasis,for example.

Another aspect of the invention is a method of inhibiting tumor growth.An effective amount of an isolated molecule comprising an antibodyvariable region which specifically binds to an extracellular domain of aTEM protein selected from the group consisting of: 1, 3, 9, 17, 19, 22,and 44, as shown in SEQ ID NO: 196, 200, 212, 230, 232, 238, and 271,respectively, is administered to a human subject bearing a tumor. Thegrowth of the tumor is consequently inhibited.

Still another aspect of the invention provides an isolated moleculecomprising an antibody variable region which specifically binds to a TEMprotein selected from the group consisting of: 3, 9, 17, 19, and 44, asshown in SEQ ID NO: 200, 212, 230, 232, and 271, respectively. Themolecule can be, for example, an in tact antibody molecule, a singlechain variable region (ScFv), a monoclonal antibody, a humanizedantibody, or a human antibody. The molecule can optionally be bound to acytotoxic moiety, bound to a therapeutic moiety, bound to a detectablemoiety, or bound to an anti-tumor agent.

According to still another aspect of the invention an isolated andpurified human transmembrane protein is provided. The protein isselected from the group consisting of: TEM 3, 9, 17, and 19 as shown inSEQ ID NO: 200, 212, 230, and 232, respectively.

Yet another aspect of the invention is an isolated and purified nucleicacid molecule comprising a coding sequence for a transmembrane TEMselected from the group consisting of: TEM 3, 9, 17, and 19 as shown inSEQ ID NO: 200, 212, 230, and 232, respectively. The isolated andpurified nucleic acid molecule may optionally comprise a coding sequenceselected from those shown in SEQ ID NO: 199, 211, 229, and 231.

Still another aspect of the invention is a recombinant host cell whichcomprises a nucleic acid molecule. The nucleic acid molecule comprises acoding sequence for a transmembrane TEM selected from the groupconsisting of: TEM 3, 9, 17, and 19 as shown in SEQ ID NO: 200, 212,230, and 232, respectively. The recombinant host cell optionallycomprises a coding sequence selected from those shown in SEQ ID NO: 199,211, 229, and 231.

According to one embodiment of the invention a method is provided forinducing an immune response in a mammal. A nucleic acid moleculecomprising a coding sequence for a human transmembrane protein selectedfrom the group consisting of: TEM 1, 3, 9, 13, 17, 19, 22, 30, and 44 asshown in SEQ ID NO: 196, 200. 212, 220, 230, 232, 238, 250, and 271,respectively, is administered to the mammal. An immune response to thehuman transmembrane protein is thereby induced in the mammal. Optionallythe coding sequence is shown in SEQ ID NO: 196, 200, 212, 220, 230, 232,238, 250 and 271.

According to yet another embodiment of the invention a method ofinducing an immune response in a mammal is provided. A purified humantransmembrane protein selected from the group consisting of: TEM 1, 3,9, 13, 17, 19, 22, 30, and 44 as shown in SEQ ID NO: 196, 200, 212, 220,230, 232, 238, 250 and 271, respectively, is administered to the mammal.An immune response to the human transmembrane protein is thereby inducedin the mammal.

Another aspect of the invention is a method for identification of aligand involved in endothelial cell regulation. A test compound iscontacted with an isolated and purified human trasmembrane proteinselected from the group consisting of 1, 3, 9, 13, 17, 30, 19, and 44 asshown in SEQ ID NO: 196, 200, 212, 220, 230, 232, 250, and 271. Theisolated and purified human trasmembrane protein is also contacted witha molecule comprising an antibody variable region which specificallybinds to an extracellular domain of a TEM protein selected from thegroup consisting of: 1, 3, 9, 13, 17, 30, 19, and 44 as shown in SEQ IDNO: 196, 200, 212, 220, 230, 232, 250, and 271 respectively. Binding ofthe molecule comprising an antibody variable region to the humantransmembrane protein is determined. A test compound which diminishesthe binding of the molecule comprising an antibody variable region tothe human transmembrane protein is identified as a ligand involved inendothelial cell regulation.

Yet another aspect of the invention is a method for identification of aligand involved in endothelial cell regulation. A test compound iscontacted with a cell comprising a human transmembrane protein selectedfrom the group consisting of 1, 3, 9, 17, and 19 as shown in SEQ ID NO:196, 200, 212, 230, and 232. The cell is also contacted with a moleculecomprising an antibody variable region which specifically binds to anextracellular domain of a TEM protein selected from the group consistingof: 1, 3, 9, 17, and 19 as shown in SEQ ID NO: 196, 200, 212, 230, and232, respectively. Binding of the molecule comprising an antibodyvariable region to the cell is determined. A test compound whichdiminishes the binding of the molecule comprising an antibody variableregion to the cell is identified as a ligand involved in endothelialcell regulation.

Yet another aspect of the invention is a method for identification of aligand involved in endothelial cell regulation. A test compound iscontacted with a human transmembrane protein selected from the groupconsisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19,20, 21, 22, 24, 25, 27, 28, 29, 40, 31, 33, 35, 36, 37, 38, 39, 41, 42,44, 45, and 46 as shown in SEQ ID NO: 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 223 & 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 358, 257, 259, 261, 263, 267,269, 271, 273, and 275. Binding of a test compound to the humantransmembrane protein is determined. A test compound which binds to theprotein is identified as a ligand involved in endothelial cellregulation.

Another embodiment of the present invention is a soluble form of a humantransmembrane protein selected from the group consisting of: TEM 1, 3,9, 17, 19, 22, 30, and 44 as shown in SEQ ID NO: 196, 200, 212, 230,232, 238, 250, and 271 respectively. The soluble forms lacktransmembrane domains. The soluble form may consist of an extracellulardomain of the human transmembrane protein.

Also provided by the present invention is a method of inhibitingneoangiogenesis in a patient. A soluble form of a human transmembraneprotein is adminstered to the patient. Neoangiogenesis in the patient isconsequently inhibited. The patient may bear a vascularized tumor, mayhave polycystic kidney disease, may have diabetic retinopathy, may haverheumatoid arthritis, or may have psoriasis, for example.

Another embodiment of the invention provides a method of inhibitingneoangiogenesis in a patient. A soluble form of a human transmembraneprotein is administered to the patient. Neoangiogenesis in the patientis consequently inhibited. The patient may bear a vascularized tumor,may have polycystic kidney disease, may have diabetic retinopathy, mayhave rheumatoid arthritis, or may have psoriasis, for example.

According to still another aspect of the invention a method ofidentifying regions of neoangiogenesis in a patient is provided. Amolecule comprising an antibody variable region which specifically bindsto an extracellular domain of a TEM protein selected from the groupconsisting of: 1, 3, 9, 13, 17, 19, 22, 30, and 44, as shown in SEQ IDNO: 196, 200, 212, 220, 230, 232, 238, 250, and 271, respectively, isadministered to a patient. The molecule is bound to a detectable moiety.The detectable moiety is detected in the patient, thereby identifyingneoangiogenesis.

According to another aspect of the invention a method is provided forinducing an immune response to tumor endothelial cells in a patient. Amouse TEM protein selected from the group consisting of: 1, 2, 3, 9, 13,17, 19, 22, and 30 as shown in SEQ ID NO: 291, 293, 299, 295, 303, 297,301, 305, and 307, is administered to a patient in need thereof. Animmune response to a human TEM protein is consequently induced.

Still another embodiment of the invention is a method of screening forneoangiogenesis in a patient. A body fluid collected from the patient iscontacted with a molecule comprising an antibody variable region whichspecifically binds to an extracellular domain of a TEM protein selectedfrom the group consisting of: 1, 3, 9, 17, 19, and 44, as shown in SEQID NO: 196, 200, 212, 230, 232, and 271, respectively. Detection ofcross-reactive material in the body fluid with the molecule indicatesneo-angiogenesis in the patient.

Still another embodiment of the invention provides a method ofinhibiting neoangiogenesis in a patient. A molecule comprising anantibody variable region which specifically binds to a TEM proteinselected from the group consisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27,31, 36, 37, 38, 39, and 40 as shown in SEQ ID NO: 202, 206, 208, 214,218, 223 and 224, 234, 242, 244, 252, 257, 259, 261, 263, and 265, isadministered to the patient. Neoangiogenesis in the patient consequentlyinhibited.

Yet another aspect of the invention is a method of screening forneoangiogenesis in a patient. A body fluid collected from the patient iscontacted with a molecule comprising an antibody variable region whichspecifically binds to a TEM protein selected from the group consistingof: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31, 36, 37, 38, 39, and 40, asshown in SEQ ID NO: 202, 206, 208, 214, 218, 223 & 224, 234, 242, 244,252, 257, 259, 261, 263, and 265, respectively. Detection ofcross-reactive material in the body fluid with the molecule indicatesneoangiogenesis in the patient.

Also provided by the present invention is a method of promotingneoangiogenesis in a patient. A TEM protein selected from the groupconsisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31, 36, 37, 38, 39, and40, as shown in SEQ ID NO: 202, 206, 208, 214, 218, 223 & 224, 234, 242,244, 252, 257, 259, 261, 263, and 265, is administered to a patient inneed of neoangiogenesis. Neoangiogenesis in the patient is consequentlystimulated.

One embodiment of the invention provides a method of promotingneoangiogenesis in a patient. A nucleic acid molecule encoding a TEMprotein selected from the group consisting of: 4, 6, 7, 10, 12, 14, 20,25, 27, 31, 36, 37, 38, 39, and 40, as shown in SEQ ID NO: 201, 205,207, 213, 217, 221 & 222, 233, 241, 243, 251, 256, 258, 260, 262, and264, is administered to a patient in need of neoangiogenesis. The TEMprotein is consequently expressed and neoangiogenesis in the patient isstimulated.

Another embodiment of the invention provides a method of screening forneoangiogenesis in a patient. A TEM protein selected from the groupconsisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31, 36, 37, 38, 39, and40, as shown in SEQ ID NO: 202, 206, 208, 214, 218, 223 & 224, 234, 242,244, 252, 257, 259, 261, 263, and 265, respectively, is detected in abody fluid collected from the patient. Detection of the TEM proteinindicates neoangiogenesis in the patient.

Another aspect of the invention is a method of screening forneoangiogenesis in a patient. A nucleic acid encoding a TEM proteinselected from the group consisting of: 4, 6, 7, 10; 12, 14, 20, 25, 27,31, 36, 37, 38, 39, and 40 is detected in a body fluid collected fromthe patient. The nucleic acid is selected from the group consisting ofthose shown in SEQ ID NO: 201, 205, 207, 213, 217, 221 & 222, 233, 241,243, 251, 256, 258, 260, 262, and 264. Detection of the TEM proteinindicates neoangiogenesis in the patient.

Yet another embodiment of the invention is an isolated and purifiednucleic acid molecule which encodes a NEM protein selected from thegroup consisting of: 14, 22, 23, and 33 as shown in SEQ ID NO: 279, 283,285, 286, 287, and 289. The nucleic acid molecule optionally comprises acoding sequence as shown in SEQ ID NO: 278, 282, 284, and 288. Thenucleic acid may be maintained in a recombinant host cell.

The present invention also provides an isolated and purified NEM proteinselected from the group consisting of: 14, 22, 23, and 33 as shown inSEQ ID NO: 279, 283, 285, 286, 287, and 289.

The present invention further provides an isolated molecule comprisingan antibody variable region which specifically binds to a NEM proteinselected from the group consisting of: 14, 22, 23, and 33, as shown inSEQ ID NO: 279, 283, 285, 286, 287, and 289.

An additional embodiment of the present invention is a method ofinhibiting neoangiogenesis. An effective amount of a NEM proteinselected from the group consisting of: 14, 22, 23, and 33 as shown inSEQ ID NO: 279, 283, 285, 286, 287, and 289 is administered to a subjectin need thereof Neoangiogenesis is thereby inhibited.

A still further embodiment of the invention is a method to identifycandidate drugs for treating tumors. Cells which express one or more TEMgenes selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 40, 31,33, 35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as shown in SEQ ID NO:195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 221 & 222,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 256, 258, 260, 262, 266, 268, 270, 272, and 274, respectively,are contacted with a test compound. Expression of said one or more TEMgenes is determined by hybridization of mRNA of said cells to a nucleicacid probe which is complementary to said mRNA. A test compound isidentified as a candidate drug for treating tumors if it decreasesexpression of said one or more TEM genes. Optionally the cells areendothelial cells. Alternatively or additionally, the cells arerecombinant host cells which are transfected with an expressionconstruct which encodes said one or more TEMs. Test compounds whichincrease expression can be identified as candidates for promoting woundhealing.

Yet another embodiment of the invention is a method to identifycandidate drugs for treating tumors. Cells which express one or more TEMproteins selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 40,31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as shown in SEQ IDNO: 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 223 &224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 358, 257, 259, 261, 263, 267, 269, 271, 273, and 275,respectively, are contacted with a test compound. The amount of said oneor more TEM proteins in said cells is determined. A test compound isidentified as a candidate drug for treating tumors if it decreases theamount of one or more TEM proteins in said cells. Optionally the cellsare endothelial cells. Alternatively or additionally, the cells arerecombinant host cells which are transfected with an expressionconstruct which encodes said one or more TEMs. Alternatively, a testcompound which increases the amount of one or more TEM proteins in saidcells is identified as a candidate drug for treating wound healing.

According to another aspect of the invention a method is provided toidentify candidate drugs for treating tumors. Cells which express one ormore TEM proteins selected from the group consisting of: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28,29, 40, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as shown inSEQ ID NO: 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,223 & 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 358, 257, 259, 261, 263, 267, 269, 271, 273, and 275,respectively, are contacted with a test compound. Activity of said oneor more TEM proteins in said cells is determined. A test compound isidentified as a candidate drug for treating tumors if it decreases theactivity of one more TEM proteins in said cells. Optionally the cellsare endothelial cells. Alternatively or additionally, the cells arerecombinant host cells which are transfected with an expressionconstruct which encodes said one or more TEMs. Optionally the cells areendothelial cells. If a test compound increases the activity of one moreTEM proteins in said cells it can be identified as a candidate drug fortreating wound healing.

An additional aspect of the invention is a method to identify candidatedrugs for treating patients bearing tumors. A test compound is contactedwith recombinant host cells which are transfected with an expressionconstruct which encodes one or more TEM proteins selected from the groupconsisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19,20, 21, 22, 24, 25, 27, 28, 29, 40, 31, 33, 35, 36, 37, 38, 39, 41, 42,44, 45, and 46 as shown in SEQ ID NO: 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 223 & 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 358, 257, 259, 261, 263, 267, 269,271, 273, and 275, respectively. Proliferation of said cells isdetermined. A test compound which inhibits proliferation of said cellsis identified as a candidate drug for treating patients bearing tumors.A test compound which stimulates proliferation of said cells isidentified as a candidate drug for promoting neoangiogenesis, such asfor use in wound healing.

Another embodiment of the invention provides a method to identifycandidate drugs for treating tumors. Cells which express one or more NEMgenes selected from the group consisting of: 14, 22, 23, and 33 as shownin SEQ ID NO: 278, 282, 284, and 288, respectively, are contacted with atest compound. Expression of said one or more NEM genes is determined byhybridization of mRNA of said cells to a nucleic acid probe which iscomplementary to said mRNA. A test compound is identified as a candidatedrug for treating tumors if it increases expression of said one or moreNEM genes. Optionally the cells are endothelial cells. Alternatively oradditionally, the cells are recombinant host cells which are transfectedwith an expression construct which encodes said one or more NEMs.

According to another aspect of the invention a method is provided toidentify candidate drugs for treating tumors. Cells which express one ormore NEM proteins selected from the group consisting of: 14, 22, 23, and33 as shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289, arecontacted with a test compound. The amount of said one or more NEMproteins in said cells is determined. A test compound is identified as acandidate drug for treating tumors if it increases the amount of onemore NEM proteins in said cells. Optionally the cells are endothelialcells. Alternatively or additionally, the cells are recombinant hostcells which are transfected with an expression construct which encodessaid one or more NEMs.

An additional aspect of the invention is a method to identify candidatedrugs for treating tumors. Cells which express one or more NEM proteinsselected from the group consisting of: 14, 22, 23, and 33 as shown inSEQ ID NO: 279, 283, 285, 286, 287, and 289, are contacted with a testcompound. Activity of said one or more NEM proteins in said cells isdetermined. A test compound is identified as a candidate drug fortreating tumors if it increases the activity of said one or more NEMproteins in said cells. Optionally the cells are endothelial cells.Alternatively or additionally, the cells are recombinant host cellswhich are transfected with an expression construct which encodes saidone or more NEMs.

Still another embodiment of the invention provides a method to identifycandidate drugs for treating patients bearing tumors. A test compound iscontacted with recombinant host cells which are transfected with anexpression construct which encodes one or more NEM proteins selectedfrom the group consisting of 14, 22, 23, and 33 as shown in SEQ ID NO:279, 283, 285, 286, 287, and 289. Proliferation of said cells isdetermined. A test compound which stimulates proliferation of said cellsis identified as a candidate drug for treating patients bearing tumors.

Another aspect of the invention is a method for identifying endothelialcells. One or more antibodies which bind specifically to a TEM or NEMprotein selected from the group consisting of TEM: 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 30,31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as shown in SEQ IDNO: 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 223 & 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 358, 257, 259, 261, 263, 267, 269, 271, 273, and 275 and NEM 14,22, 23, and 33 as shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289,is contacted with a population of cells. Cells in the population whichhave bound to said antibodies are detected. Cells which are bound tosaid antibodies are identified as endothelial cells. Optionally cellswhich have bound to said antibodies are isolated from cells which havenot bound.

Still another aspect of the invention is a method for identifyingendothelial cells. One or more nucleic acid hybridization probes whichare complementary to a TEM or NEM gene nucleic acid sequence selectedfrom the group consisting of TEM: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 30, 31, 33, 35, 36,37, 38, 39, 41, 42, 44, 45, and 46 as shown in SEQ ID NO: 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 223 & 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 358, 257, 259,261, 263, 267, 269, 271, 273, and 275 and NEM 14, 22, 23, and 33 asshown in SEQ ID NO: 279, 283, 285, 286, 287, and 289, is contacted withnucleic acids of a population of cells. Nucleic acids which havespecifically hybridized to said nucleic acid hybridization probes aredetected. Cells whose nucleic acids specifically hybridized areidentified as endothelial cells.

Yet another embodiment of the invention is a method of inhibitingneoangiogenesis. An effective amount of an isolated molecule comprisingan antibody variable region which specifically binds to an extracellulardomain of a mouse TEM protein selected from the group consisting of: 1,2, 3, 9, 17, and 19, as shown in SEQ ID NO: 291, 293, 299, 295, 297, and301, respectively, is administered to a subject in need thereof.Neoangiogenesis is thereby inhibited. The subject may be a mouse, maybear a vascularized tumor, may have polycystic kidney disease, may havediabetic retinopathy may have rheumatoid arthritis, or may havepsoriasis, for example.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with reagents andmethods for detection, diagnosis, therapy, and drug screening pertainingto neoangiogenesis and pathological processes involving or requiringneoangiogenesis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. vWF expression in colorectal cancers. vWF (red stain) wasdetected in vessels by in situ hybridization. At low power magnification(FIG. 1.A) vessels were often surrounded by a perivascular cuff ofviable cells (red arrows), with a ring of necrotic cells evident at theperiphery (black arrows). At high power magnification (FIG. 1.B) theexpression of vWF (red) was clearly localized to the vessels. Sectionswere counterstained with methyl green.

FIG. 2A-2D. Purification of Endothelial Cells (ECs) from human normaland malignant tissue. (FIG. 2A) Vessels (red) of frozen sections werestained by immunofluorescence with the P1H12 monoclonal antibody(Chemicon, Temecula, Calif.) and detected using a biotinylated goatanti-mouse IgG secondary antibody followed by rhodamine-linkedstrepavidin. The region stained is from within the lamina propria ofnormal colonic mucosa. Note that the larger vessels (arrowheads) andcapillaries (arrows) are positive, and staining of hematopoietic cellswas undetectable. E-cadherin positive epithelial cells (green) at theedge of the crypt were simultaneously visualized using a rabbitpolyclonal antibody (Santa Cruz, Santa Cruz, Calif.), followed by a goatanti-rabbit IgG secondary antibody labelled with alexa (MolecularProbes, Eugene, Oreg.). Sections were imaged at 60× magnification usingconfocal microscopy. (FIG. 2.B) To isolate pure populations fromcollagenase dispersed tissues, the epithelial and hematopoietic cellfractions were sequentially removed by negative selection with magneticbeads. The remaining cells were stained with P1H12 and ECs were isolatedby positive selection with magnetic beads. (FIG. 2.C) RT-PCR analysisused to assess the purity of the EC preparations. Semiquantitative PCRanalysis was performed on cDNA generated either directly from colorectalcancer tissue (unfractionated tumor) or from purified ECs isolated fromnormal colonic mucosa (normal EC fraction) or colorectal cancer (tumorEC fraction). PCR amplification of the epithelial specific markercytokeratin 20 (CK20), demonstrated its expression was limited to theunfractionated tumor. Two endothelial specific markers, vWF andVE-cadherin (VE-Cad) showed robust amplification only in the endothelialfractions, validating the purity and enrichment protocol shown in (FIG.2.B). The ubiquitous housekeeping enzyme GAPDH was observed in allsamples. No signal was detected in the no-template (NT) control. cDNAtemplates were diluted 1:10, 1:100, 1:1000, 1:4000, and 1:40,000 asindicated by the declining wedge. (FIG. 2.D) The relative expressionlevel of select genes was determined by measuring the tag abundance fromseveral SAGE libraries combined into four groups. The first was composedof ˜193,000 tags from the two in vivo-derived EC preparations(Endothelial Cell Fraction) while the second contained a single libraryof ˜57,000 tags containing macrophages and other leukocytes derived fromthe negative selection (Hematopoietic Fraction). The fourth librarycontained ˜401,000 tags from cultured HUVEC and HMVEC (Endothelial Cellsin Culture), and the fourth consisted of ˜748,000 tags from 6 coloncancer cell lines in culture (Epithelial Cells). After normalization,the library with the highest tag number for each marker was given avalue of 100%, and the corresponding relative expression levels of theremaining 3 libraries was plotted on the ordinate. Note the high levelof CD31 present on hematopoietic cells, the likely cause of the impurityof the initial endothelial selection, compared with the selectivity ofP1H12.

FIG. 3A-3E). Expression of Pan-Endothelial Markers (PEMs) is limited toECs. The endothelial origin of PEMs identified by SAGE was confirmedusing a highly sensitive in situ hybridization assay. Localization ofnovel PEMs to the ECs was demonstrated by examining two representativePEMs, PEM3 (FIG. 3A) and PEM6 (FIG. 3B) in lung cancer and colon cancer,respectively. Hevin expression was readily detected in the ECs of acolon tumor (FIG. 3C) despite its low level of expression in culturedECs. Expression of VEGFR2 was readily detectable in the ECs of bothnormal (FIG. 3D) and malignant colon tissue (FIG. 3E).

FIG. 4A-4J. Expression of Tumor Endothelial Markers (TEMs). (FIG. 4A)RT-PCR analysis confirmed the tumor specific expression of selectednovel TEMs. Semiquantitative PCR analysis was performed on cDNAgenerated either from purified epithelial cells as a negative control(Control) or from purified ECs isolated from normal colonic mucosa(Normal ECs) or colorectal cancer (Tumor ECs) from two differentpatients. Two endothelial specific markers, vWF and PEM6 showed robustamplification only in the endothelial fractions whereas the ubiquitoushousekeeping enzyme GAPDH was observed in all samples. TEM1 (BSC-TEM1),TEM 17 (BSC-TEM7) and TEM22 (BSC-TEM9) were specifically expressed intumor compared to normal ECs. The cDNA template was diluted 1:10, 1:100,1:1000, and 1:10,000 as indicated by the declining wedge. (FIG. 4B-4J)The endothelial origin of TEMs identified by SAGE was confirmed using insitu hybridization as in FIG. 3. Expression of TEM 1 (BSC-TEM1) (FIG.4B) and TEM17 (BSC-TEM7). (FIG. 4C) was demonstrated to be highlyspecific to the ECs in colorectal cancers; sections were imaged in theabsence of a counterstain to show the complete lack of detectableexpression in the non-endothelial cells of the tumor. Expression ofTEM17 (BSC-TEM7) in ECs was demonstrated in a metastatic liver lesionfrom a primary colorectal cancer (FIG. 4D), a lung (FIG. 4E), breast(FIG. 4F), pancreatic (FIG. 4G) and brain cancer (FIG. 4H), as well asin a sarcoma (FIG. 41). TEM 17 (BSC-TEM7) was also localized to vesselsduring normal physiological angiogenesis of the corpus luteum (FIG. 4J).

DETAILED DESCRIPTION OF THE INVENTION

We identified 46 human genes that were expressed at significantly higherlevels (>10-fold) in tumor endothelium than in normal endothelium, and33 genes that were expressed at significantly lower levels in humantumor versus normal endothelium. See Tables 2 and 4, respectively. Mostof these genes were either not expressed or expressed at relatively lowlevels in Endothelial Cells (ECs) maintained in culture. Moreover, weidentified 93 genes which are expressed in both normal and tumor humanendothelium. Interestingly, the tumor endothelium genes were expressedin all tumors tested, regardless of its tissue or organ source. Mosttumor endothelium genes were also expressed in corpus luteum and wounds.

As the work has progressed, we have refined and classified our original46 tumor endothelial markers. We have named these markers TEMs andrenumbered them consecutively by the prevalence of their tags in ourSAGE analysis. Originally we had not used a consecutive numberingsystem. Our non-consecutive numbering system has been renamed asBSC-TEMs. For most of the original 46 SAGE Tags, we now providefull-length nucleic acid and protein sequence. In some cases, thesequences were obtained through the public databases, in others thesequences were obtained by cloning and through the use of geneprediction tools. In some cases, we found SAGE Tags corresponding togenes having different splice varients or with known polymorphisms. Forexample, in one case the SAGE Tag BSC-TEM3 has been found to hybridizeto an alternatively spliced form of the transcript encoding BSC-TEM7.The proteins encoded by the two transcripts are the same; therefore theyare cumulatively called TEM7. A highly related sequence was found viahomology searches, BSC-TEM7R. This paralog sequence is now called TEM3.See Table 2, which follows, showing tumor endothelial markers by orderof prevalence (except for TEM 3). Column 1 indicates the prevalencenumber. Column 2 indicates the original nomenclature. Column 3 indicatesthe short tags. Column 4 indicates the long tags. Column 5 indicates theaccession number in GenBank. Column 6 indicates the sequence identifiersfor the short tag, the long tag, the full nucleic acid, and the protein.Column 7 provides a functional description, which is expanded below inthe text.

TEM BSC- GGGGCTGCC GGGGCTGCCCAGCT NM020404 SEQ ID tumor endothelialmarker 1 precursor 1 TEM1 CA GA NO: 94, 309, 195, 196 TEM BSC- GATCTCCGTSEQ ID sapiens tumor endothelial marker 2 2 TEM2 GT NO: 95, (BSC-TEM2)mRNA/mouse Ras, dexa- 197, 198 methasone-induced 1 (RASD1), mRNA TEMBSC- SEQ ID human ortholog of mouse paralog of 3 TEM7 N0: 199, mouseTEM-7 R 200 TEM CTTTCTTTGA CTTTCTTTGAGTTTT AB034203 SEQ ID Homo sapiensdickkopf-3 (DKK-3) 4 G AA NO: 97, mRNA, 311, 201, 202 TEM BSC-TATTAACTCT TATTAACTCTCTTTG SEQ ID Tumor endothelial marker 4 5 TEM4 C GAN0: 98, 312, 203, 204 TEM CAGGAGACC CAGGAGACCCCAGG X57766 SEQ ID Humanstromelysin-3 mRNA. 6 CC CCC NO: 99, 314, 205, 206 TEM GGAAATGTCGGAAATGTCAGCAA BC002576 SEQ ID matrix metalloproteinase 2 7 AA GTA NO:100, (gelatinase A, 72kD gelatinase, 72kD 315, 207, type IV collagenase)208 TEM CCTGGTTCA SEQ ID HeyL transcription factor 8 GT NO: 101, 316,209, 210 TEM BSC- TTTTTAAGAA TTTTTAAGAACTCGG SEQ ID 9 TEM5 C GT NO: 102,317, 211, 212 TEM TTTGGTTTTC TTTGGTTTTCCAAAA J03464, SEQ ID Humancollagen alpha-2 type I mRNA, 10 C GA M18057, NO: 103, complete cds,clone pHCOL2A1. X02488 319, 213, 214 TEM ATTTTGTATG ATTTTGTATGATTTTNM_00250 SEQ ID nidogen/entactin 11 A TA 8 NO: 104, 321, 215, 216 TEMACTTTAGATG ACTTTAGATGGGAA X52022 SEQ ID H. sapiens RNA for type VIcollagen 12 G GCC NO: 105, alpha3 chain. 322, 217, 218 TEM GAGTGAGACGAGTGAGACCCAGG M11749 SEQ ID Human Thy-1 glycoprotein gene, 13 CC AGCNO: 106, complete cds. 324, 219, 220 TEM GTACACACA GTACACACACCCCC SEQ IDCystatin SN 14 CC ACC NO: 107, 325, 221, 223 TEM GTACACACAGTACACACACCCCC X54667 SEQ ID H.sapiens mRNA for cystatin S. 14 CC ACCNO: 107, 325, 222 224 TEM CCACAGGGG CCACAGGGGATTCT NM_00009 SEQ ID HumanmRNA 3′ region for pro-alpha1 15 AT CCT 0 NO: 108, (III) collagen. 327,225 226 TEM BSC- TTAAAAGTCA TTAAAAGTCACTGTG SEQ ID 16 TEM6 C CA NO: 109,328, 227, 228 TEM BSC- ACAGACTGTT ACAGACTGTTAGCC AF279144 SEQ ID HumanTumor endothelial marker 7 17 TEM7 A AAG NO: 110, 329, 229, 230 TEMCCACTGCAA SEQ ID 18 CC NO: 111 TEM BSC- CTATAGGAG SEQ ID 19 TEM8 AC NO:112, 330, 231, 232 TEM GTTCCACAG NM_00008 SEQ ID collagen, type I, alpha2 (COL1A2 20 AA 9 NO: 113, 233, 234 TEM TACCACCTC TACCACCTCCCTTTC SEQ IDHomo sapiens mRNA; cDNA DKFZp762B245 21 CC CT NO: 114, (from cloneDKFZp762B245); 331, 235, 236 TEM BSC- GCCCTTTCTC GCCCTTTCTCTGTA NM_00603SEQ ID endocytic receptor (macrophage 22 TEM9 T GTT 9 NO: 115, mannosereceptor family) (KIAA0709), 334, 237, 238 TEM TTAAATAGCATTAAATAGCACCTTT SEQ ID no match 23 C AG NO: 116, 335 TEM AGACATACTAGACATACTGACAG NM_02264 SEQ ID Homo sapiens mRNA; cDNA DKFZp434G162 24GA AAT 8 NO: 117, (from clone DKFZp434G162); 336, 239, 240 TEM TCCCCCAGGTCCCCCAGGAGCCA L35279, SEQ ID Homo sapiens (clone kT2) bone 25 AG CCGNM_00612 NO: 118, morphogenetic protein-1 (BMP-1) mRNA 9 338, 241, 242TEM AGCCCAAAG SEQ ID No Match 26 TG NO: 119 TEM ACTACCATAA NM_00306 SEQID Homo sapiens mRNA for MEGF5, partial 27 C 2 NO: 120, cds. 243, 244TEM TACAAATCGT TACAAATCGTTGTCA NM_01485 SEQ ID Homo sapiens mRNA forKIAA0672 28 T AA 9 NO: 121, protein, complete cds. 339, 245, 246 TEMTTGGGTGAA SEQ ID ESTs (2 unigene clusters) 29 AA NO: 122, 247, 248 TEMCATTATCCAA CATTATCCAAAAACA THC53402 SEQ ID integrin, alpha 1 30 A AT 9,NO: 123, X68742, 340, 249, AI262158, 250 AI88747, AI394565, AA679721 TEMAGAAACCAC AGAAACCACGGAAA NM_00184 SEQ ID hypothetical protein KIAA116431 GG TGG 5 NO: 124, 341, 251 252 TEM ACCAAAACC SEQ ID no match 32 ACNO: 125 TEM TGAAATAAAC NM_00025 SEQ ID methylmalonyl Coenzyme A mutase33 5 NO: 126, 253, 254 TEM TTTGGTTTCC SEQ ID no match 34 NO: 127 TEMGTGGAGACG GTGGAGACGGACTC ESTAI186 SEQ ID est 35 GA TGT 535 N0: 128 345,255, 358 TEM TTTGTGTTGT TTTGTGTTGTATATT NM_00437 SEQ ID est 36 A TA 0NO: 129, 346, 256, 257 TEM TTATGTTTAA TTATGTTTAATAGTT NM_00234 SEQ IDHuman lumican mRNA, complete cds. 37 T GA 5 NO: 130, 347, 258, 259 TEMTGGAAATGA TGGAAATGACCCAA NM_00008 SEQ ID collagen type1 alpha1 38 C AAA8 NO: 131, 348, 260, 261 TEM TGCCACACA TGCCACACAGTGAC NM_00323 SEQ IDHuman transforming growth factor- 39 GT TTG 9 NO: 132, beta 3(TGF-beta3) mRNA, complete 350, 262, 263 TEM GATGAGGAG GATGAGGAGACTGGSEQ ID collagen, type I, alpha 2 40 AC CAA NO: 133, 351, 264, 265 TEMATCAAAGGTT ATCAAAGGTTTGATT SEQ ID est 41 T TA N0: 134, 352, 266, 267 TEMAGTCACTAGT AGTCACATAGTACAT NM_02522 SEQ ID ESTs 42 AA 6 NO: 135, 353,268, 269 TEM TTCGGTTGG TTCGGTTGGTCAAA SEQ ID No match 43 TC GAT NO: 136,354 TEM CCCCACACG CCCCACACGGGCAA NM_01835 SEQ ID Homo sapiens cDNAFLJ11190 fis, 44 GG GCA 4v NO: 137, clone PLACE1007583. 355, 270, 271TEM GGCTTGCCT GGCTTGCCTTTTTGT NM_00036 SEQ ID est 45 TT AT 6 NO: 138,356, 272, 273 TEM ATCCCTTCCC ATCCCTTCCCGCCA NM_00268 SEQ ID Homo sapiensmRNA for peanut-like 46 G CAC 8 NO: 139, protein 1, PNUTL1 (hCDCrel-1).357, 274, 275

The studies described below provide the first definitive molecularcharacterization of ECs in an unbiased and general manner. They lead toseveral important conclusions that have direct bearing on long-standinghypotheses about angiogenesis. First, it is clear that normal and tumorendothelium are highly related, sharing many endothelial cell specificmarkers. Second, it is equally clear that the endothelium derived fromtumors is qualitatively different from that derived from normal tissuesof the same type and is also different from primary endothelialcultures. Third, these genes are characteristically expressed in tumorsderived from several different tissue types, documenting that tumorendothelium, in general, is different from normal endothelium. Fourth,the genes expressed differentially in tumor endothelium are alsoexpressed during other angiogenic processes such as corpus luteumformation and wound healing. It is therefore more appropriate to regardthe formation of new vessels in tumors as “neoangiogenesis” rather than“tumor angiogenesis” per se. This distinction is important from avariety of perspectives, and is consistent with the idea that tumorsrecruit vasculature using much of, or basically the same signalselaborated during other physiologic or pathological processes. Thattumors represent “unhealed wounds” is one of the oldest ideas in cancerbiology.

The nature and precise biological function of many of the TumorEndothelial Markers (TEMs) identified here are unknown. Of thepreviously characterized genes shown in Table 2, it is intriguing thatseveral encode proteins involved in extracellular matrix formation orremodelling (TEM 6, TEM 6, TEM 10, TEM 7, TEM 11, TEM 12, TEM 14, TEM20, TEM 24, TEM 25, TEM 27, TEM 37, TEM 38, and TEM 40,) Deposition ofextracellular matrix is likely critical to the growth of new vessels.Finally, it is perhaps not surprising that so many of theendothelial-specific transcripts identified here, whether expressed onlyin neovasculature or in endothelium in general, have not been previouslycharacterized, and some are not even represented in EST databases. Inpart, this may be due to the fact that the EST databases are heavilybiased toward certain tissues, but moreover, may be due to the fact thateven in highly vascularized tissues endothelial cells are still arelatively small proportion of the population. Thus, the sensitivity ofthe SAGE method is a particularly appropriate tool.

Sequence and literature study has permitted the followingidentifications to be made among the family of TEM proteins. TEMproteins have been identified which contain transmembrane regions. Theseinclude TEM 1, TEM 3, TEM 9, TEM 13, TEM 17, TEM 19, TEM 22, TEM 30, andTEM 44. TEM proteins have been identified which are secreted proteins,including TEM 4, TEM 6, TEM 7, TEM 10, TEM 12, TEM 14, TEM 20, TEM 25,TEM 27, TEM 31, TEM 36, TEM 37, TEM 38, and TEM 39. HeyL (TEM 8) is atranscription factor which may be involved in regulating TEMs as one ormore groups. The protein corresponding to the tag for TEM44 was found inthe public databases, but no biological function has yet been ascribedto it.

TEM 1 has been named endosialin in the literature. It has a signalsequence at amino acids 1-17 and a transmembrane domain at amino acids686-708. Thus it is a cell surface protein. Its extracellular domain isat resiudes 1-685. Endosialin may be involved in endocytosis. The mouseortholog is predicted to have a signal peptide at residues 1-21.

TEM 2 is a dexamethasone induced, as related protein homolog of 266amino acids. It has neither a signal sequence nor a transmembranedomain. Thus it is neither a cell surface nor a secreted protein. TEM 2plays a role in signal transduction. It regulates alterations in cellmorphology, proliferation, and cell-extracellular matrix interactions.

TEM 3 (originally termed TEM 7R) has both a signal sequence (at residues1-24 or 1-30) and a transmembrane domain (at residues 456-477). Thus itis a cell surface protein. The portion of the protein which isextracelular is at amino acids 1-455. TEM 3 has domains with homology tointegrins, plexin, and adhesion molecules. TEM 3 may regulate GTPasesthat control signal transduction pathways linking plasma membranereceptors to the actin cytoskeleton. In the mouse ortholog, the signalpeptide is predicted to be residues 1-30.

TEM 4 is also known as DKK-3. It has a signal sequence (residues1-16),suggesting that is a secreted protein. TEM 4 regulates wnt signaling,and it may be involved in vasculogenesis and wnt-dependent signaling forendothelial growth. TEM 4 is an inhibitor of Wnt oncogene and suchinhibition can be determined by assay. Tsuji et al., Biochem. Biophys.Res. Comm. 268: 20-4, 2000.

TEM 5 appears to be neither secreted nor a cell surface protein. TEM 5appears to be a component of a G protein—GTPase signaling pathway.

TEM 6 is also known as stromelysin-3/Matrix metalloproteinase 11(MMP-11). It has a signal sequence at residues 1-31, but notransmembrane domain. It has an alternative signal peptide splice siteat residues 108-109. Thus it appears to be a secreted protein. TEM 6belongs to the zinc metaloprotease family, also known as the matrixinsubfamily. TEM 6 is expressed in most invasive carcinomas. Alpha1—protease inhibitor is a natural substrate of MMP 11. TEM 6 degradesextracellular matrix proteins such as collagen and is involved inextracellular matrix remodeling and cell migration. Stromelysin can beassayed using a casein-resorufin substrate, for example. See Tortorellaand Arner, Inflammation Research 46 Supp. 2: S122-3, 1997.

TEM 7 is a protein of many names, also being known as matrixmetalloproeinase 2, gelatinase A, and 72 KD type IV collagenase. TEM 7has a signal sequence at residues 1-26 and is a secreted protein. LikeTEM 6, TEM 7 belongs to the matrixin subfamily (zincmetalloproteinases). TEM 7 cleaves gelatin type I, collagen type I, IV,V VII and X. TEM 7 associates with integrin on the surface ofendothelial cells and promotes vascular invasion. TEM 7 is involved intissue remodeling. TEM 7 can be assayed using zymography or quenchedfluorescent substrate hydrolysis, for example. Garbett, et al.,Molecular Pathology 53: 99-106, 2000. A fluorogenic matrixmetalloproteinase substrae assay can also be used which employsmethoxycoumarin containing septapeptide analog of the alpha2(I) collagencleavage site. See Bhide et al., J. Periodontology 71:690-700, 2000.

TEM 8 is HEYL protein. It has neither a signal sequence nor atransmembrane domain. It is related to the hairy/Enhancer of splitgenes. TEM 8 is likely a nuclear protein, having a role as atranscription factor. TEM 8 belongs to a new class of Notch signaltranducers and plays a key role in various developmental processes, suchas vascular development, somatogenesis and neurogenesis. SNP's atresidues 615 and 2201 have Cytosine bases. Notch 3 mutations underliethe CADASIL vascular disorder. See Mech Dev 2000 November; 98 (1-2):175.

TEM 9 is a G-protein coupled receptor homolog, having both a signalsequence at residues 1-26 and 7 transmembrane domains. Thus it is a cellsurface protein. Its extracellular region resides in amino acids 1-769.Its transmembrane domains are at residues 817-829 (TM2 and TM3),residues 899-929 (TM4 and TM5), and residues 1034-1040 (TM6 and TM7).TEM 9 acts as a G-protein coupled receptor with extracellular domainscharacteristic of cell adhesion proteins. One of its splice variants mayfunction as a soluble receptor. TEM 9 may regulate cell polarity andcell migration. It may be involved in exocytosis based on latrophilinfunction. The mouse ortholog has a predicted signal peptide at residues1-29.

TEM 10 is collagen type I, alpha2 (COL1A2), which has a signal sequenceat residues 1-22. It is an extracellular matrix (ECM) protein which issecreted subsequent to synthesis. TEM 10 interacts with a number ofproteins including other ECM proteins, certain growth factors, andmatrix metalloproteases. TEM 10 is required for the induction ofendothelial tube formation and is involved in tissue remodeling. Avariant at nucleotide 3233 which substitutes an A, is associated withosteogenesis imperfecta type IV. A variant at nucleotide 4321substituting an A retains a wild type phenotype. Nucleotide 715 is asite of a polymorphism. Nucleotides 695-748 are deleted in Ehlers-Danossyndrome. Other mutations are associated with idiopathic osteoporosis,and atypical Marfan syndrome. Variants are known at nucleotides226(T,C), 314(A,C), 385(T,C), 868 (G,A), 907(C,T), 965(A,G), 970(T,A),1784 (G,C), 2017(T,G), 2172(C,A), 2284(T,C), 2308(T,C), 2323(T,G),2344(T,G), 2604(G,A), 2974(A,T), 2903(A,G), 2995(C,T), 3274(C,T),3581(A,C), 3991(A,C), 4201(G,T), 4434(C,T), 4551(A,C), 4606(C,A),4947(T,C), 4978(C,T), 4982(G,T), 5051(G,T). PolyA sites are located atnucleotides 4450, 4550, 4885, and 5082. PolyA signals are located at4420-4424, 4515-4520, 4529-4534, 4866-4871, 5032-5037, 5053-5058. TEM10, 20, and 40 derive from the same gene but are different isoformshaving different lengths.

TEM 11 is Nidogen/Entactin. It is a secreted protein which has a signalsequence at residues 1-28. TEM 11 is an extracellular matrix proteinwhich is a component of a basement membrane. TEM 11 binds to laminin andcollagen IV and other extracellular matrix proteins. TEM 11 regulatescapillary formation and is involved in tissue remodelling. Variationshave been observed at nucleotides 4265(T,C), 4267(G,C,T), and 4738(T,G).Nidogen can be assayed by its effect on the morphology of astrocytes.See Grimpe et al., GLIA 28:138-49, 1999.

TEM 12 is the alpha 3 chain of collagen type VI. It has a signalsequence at residues 1-25. A secreted protein, TEM 12 is anextrallcellular matrix protein. TEM 12 has a splice variant. TEM 12 is amajor constituent of vascular subendothelium and is involved in tissueremodeling. It regulates platelet activation and aggregation.Alternatively spliced domains are located at nucleotides 347-964,965-1567, 2153-3752, and 4541-5041.

TEM 13 is also known as Thy-1 glycoprotein. It has both a signalsequence (at residues 1-19) and a transmembrane domain (at residues143-159). Residues 131-161 are removed in a matured form of the protein.The extracellular region of the protein is resudes 1-142 or residues1-130. TEM 13 has a glycosyl phosphatidylinositol (GPI) anchor atresidue 130 anchoring it to the membrane. TEM 13 is detectale in itssoluble form in human serum. TEM 13 is reported to be a marker foractivated endothelial cells (a marker of adult but not embryonicangiogenesis). TEM 13 on vascular endothelial cells may function as apossible vascular permeability modulator. Antibody to Thy-1 is amitogenic signal for the CD4+CD45+ and CD8+CD45+ cells, but fails toinduce proliferation in the CD45− T cells. Pingel et al., InternationalImmunology 6:169-78, 1994. Thy-1 can be assayed as an inhibitor of suchsignal.

TEM 14 is also known as cystatin S. It is a secreted protein with asignal sequence at residues 1-20 and an extracellular region at residues1-141. It is a cysteine protease inhibitor. TEM 14 may regulate cysteineprotease function involved in angiogenesis and tissue remodeling. TEM14is an inhibitor of the activity of papain and such inhibition can beassayed. Hiltke et al., J. Dental Research 78:1401-9, 1999.

TEM 15 is collagen type III, alpha 1 (COL3A1). It has a signal sequence(residues 1-23) and is secreted. Type III collagen binds to vonWillebrand factor. It is involved in cell-cell adhesion, proliferation,and migration activities. Variants at nucloetides 2104(C,A), 2194(G,A),2346(C,T), 2740(C,T), 3157(T), 3468(G), 3652(T), 3666(C), 3693(C),3755(G), 3756(T), 3824(C), 4546(A, G), 4661(G), 4591(C,T), 4665(C),5292(C), 5293(C), and 5451(A) have been observed.

TEM 16 is a tensin homolog which is apparently an intracellular protein.It may have splice variants or isoforms. One form with 1704 amino acidshas a region at the N-terminal domain which is similar to a tumorsuppressor protein, phosphatase and tensin homolog (PTEN). Tensin is afocal adhesion molecule that binds to actins and phosphorylatedproteins. It is involved in cell migration linking signal tranductionpathways to the cytoskeleton. PTEN regulates tumor induced angiogenesis.

TEM 17 (BSC-TEM 7) has a signal sequence which includes residues 1-18and a transmembrane domain at residues 427-445. It is a cell surfacemarker with an extracellular region comprising residues 1-426. It hashomologs in both mouse and C. elegans. Residues 137-244 share weakhomology with nidogen; residues 280-344 share homology to PSI domainsfound in plexin, semaphorins and integrin beta subunits. Variants havebeen observed at nucleotides 1893(A,G), 1950(C,G), 2042(A,G), and2220(G,A). In mouse TEM 17 the signal sequence includes residues 1-19.

TEM 19 was originally reported to be tumor endothelial marker 8, i.e.,BSC-TEM 8. It has a signal sequence at residues 1-27 and a transmembranedomain at residues 322-343. It is a cell surface protein having anextracellular region at residues 1-321.TEM 19 has a von WillebrandFactor (vWF) A domain at residues 44-216; a domain at residues 34-253which is found in leukointegrin alpha D chain; and a domain at residues408-560 found in PRAM-1 or adaptor molecule−1 of the vinculin family.TEM 19's function is adhesion related. vonWillibrand Factor domains aretypically involved in a variety of functions including vascularprocesses. TEM 19 may play a role in the migration of vascularendothelial cells. The mouse ortholog has a predicted signal peptide atresidues 1-27.

TEM 20 is collagen type I, alpha 2 (COL1A2). It has a signal sequence atresidues 1-22 and is a secreted extracellular matrix protein. TEM 20induces endothelial tube formation in vitro and is involved in tissueremodeling. Variants have been observed at nucleotides 226(T,C),314(A,C), 385(T,C), 868 (G,A), 907(C,T), 965(A,G), 970(T,A), 1784(G,C),2017(T,G), 2172(C,A), 2284(T,C), 2308(T,C), 2323(T,G), 2344(T,G),2604(G,A), 2794(A,T), 2903(A,G), 2995(C,T), 3274(C,T), 3581(A,C),3991(A,C), 4201(G,T), 4434(C,T), 4551(A,C), 4606(C,A), 4895-4901(—,GGACAAC), 4947(T,C), 4978(C,T), 4982(G,T), 5051(G,T).

TEM 21 is a Formin-like protein homolog which is an intracellularprotein. Formin related proteins interact with Rho family small GTPases,profilin, and other actin associated proteins. Formin-binding proteinsbind to FH1 domains with their WW domains. TEM 21 has a proline rich FH1 domain at residues 221-449. Formin related proteins play crucial rolesin morphogenesis, cell polarity, cytokinesis and reorganization of theactin cytoskeleton. They may also regulate apoptosis, cell adhesion andmigration.

TEM 22 is an endocytic receptor in the macrophage mannose receptorfamily. It has both a signal sequence at residues 1-30 and atransmembrane domain at residues 1415-1435, and resides on the cellsurface. Its extracellular domain is amino acids 1-1414. TEM 22 may bepresent as a soluble (secreted) form and act as an inhibitor. It maybind secreted phopholipase A2 (sPLA2) and mediate biological responseselicited by sPLA2. TEM 22 may have endocytic properties for sPLA2 andmediate endocytosis for endothelial related proteins. It may promotecell adhesion and be involved in cell-cell communication. Variationshave been observed at nucleotide 5389 (A, G). TEM 22 mediates uptake ofmicro-organisms and host-derived glycoproteins. Groger et al., J.Immunology 165:5428-34, 2000.

TEM 24 is tensin, an intracellular protein. It is a focal adhesionmolecule that binds to actin filaments and interacts withphosphotyrosine containing proteins. It may mediate kinase signalingactivities and regulate cellular transformation. Variations have beenobserved at nucleotides 2502 (A, G), 2622(A, G), 6027(A, G). TEM24 bindsto actin filaments and interacts with phosphotyrosine-containingproteins. Chen et al., Biochem. J. 351 Pt2:403-11, 2000. TEM24 alsobinds to phosphoinositide3-kinase. Auger et al., J. Bio. Chem.271:23452-7, 1996 TEM 24 also binds to nuclear protein p130. Lo et al.,Bioessays 16:817-23, 1994.

TEM 25 is Bone morphogenic protein 1 (BMP-1) which has a signal sequenceat residues 1-22. It is a secreted protein. There are at least 6isoforms of BMP-1 as well as splice variants which add carboxy terminalCUB domains and an additional EGF domain. TEM 25 is a metalloproteaseenzyme. It cleaves the C-terminal propeptide of collagen type I, II andIII and laminin 5 gamma 2 proteins that are important for vascularprocesses. It is involved in cartilage formation. Variations have beenobserved at nucleotides 3106(C,T), 3248(G,A), 3369(G,A). TEM 25 cleaveprobiglycan at a single site, removing the propeptide and producing abiglycan molecule with an NH(2) terminus identical to that of the matureform found in tissues. Sctt et al., J. Biol. Chem. 275:30504-11, 2000.Laminin alpha 3 and gamma2 short chains are substraates of TEM 25. Amanoet al., J. Biol. Chem. 275:22728-35, 2000.

TEM 27 is known as Slit homolog 3, a secreted protein with a signalsequence at residues 1-27. TEM 27 is a secreted guide protein involvedin migration, repulsion and patterning. It interacts with “round about”receptors (Robo receptors). TEM 27 may interact with extracellularmatrix (ECM) proteins and is involved in cell adhesion. Variations havebeen observed at nucleotides 4772 (C,T).

TEM 28 is similar to mouse nadrin (neuron specific GTPase activiatingprotein). TEM 28 is an intracellular protein with a RhoGAP domain. TheRhoGAP domain activates RhoA, Rac 1, and Cdc42 GTPases. It is involvedin the reorganization of actin filaments and enhancing exocytosis. Itmay also be involved in cell signalling. Variations have been observedat nucleotide 3969 (A,C),

TEM 29 is protein tyrosine phosphatase type IVA, member 3, isoform 1, anintracellular protein. It has alternate splice variants. TEM 29 belongsto a small class of prenylated protein tyrosine phosphatases (PTPs). Itmay be membrane associated by prenylation. PTPs are cell signalingmolecules and play regulatory roles in a variety of cellular processesand promote cell proliferation. PTP PRL-3 regulates angiotensin—IIinduced signaling events.

TEM 30 is integrin alpha 1, a cell surface protein having both a signalsequence (residues 1-28) and a transmembrane domain (residues1142-1164). Its extracellular region includes amino acids 1-1141. TEM 30is a receptor for laminin and collagen. It mediates a variety ofadhesive interactions. TEM 30 is abundantly expressed on microvascularendothelial cells. It stimulates endothelial cell proliferation andvascularization. TEM 30 may regulate angiostatin production. Variationshave been observed at nucleotide 418 (C,T). TEM 30 activates theRas/Shc/mitogen-activated protein kinase pathway promoting fibroblastcell proliferation. It also acts to inhibit collagen andmetalloproteinase synthesis. Pozzi et al., Proc. Nat. Acad. Sci. USA97:2202-7, 2000,

TEM 31 is Collagen IV alpha 1 (COL4A1) a secreted protein with a atresidues 1-27. TEM 31 is a component of the basement membrane. It bindsto alpha3 beta lintegrin and promotes integrin mediated cell adhesion.Non-collagenous domains of type IV subunits are involved in tumoralangiogenesis. TEM 31 is involved in tissue remodeling. Variations havebeen observed at nucleotide 4470 (C,T).

TEM 33 is methylmalonyl Co-A Mutase a protein which is localized in themitochondrial matrix. It degrades several amino acids, odd-numbered-acidfatty acids, and cholesterol to the tricarboylic acid cycle. A defect inTEM 33 causes a fatal disorder in organic acid metabolism termedmethylmalonic acidurea. Variations have been observed at nucleotides1531(G,A), 1671(G,A), 2028(T,C), 2087(G,A), 2359(A,G), 2437(C,A),2643(G,C), 2702(G,C). TEM 33 converts L-methylmalonyl CoA to succinylCoA. This reaction can be assayed as is known in the art. See, e.g.,Clin. Chem. 41(8 Pt I):1164-70, 1995.

TEM 36 is collagen type XII, alpha1 (COL12A1), an extracellular matrixprotein having a signal sequence at residues 1-23 or 24. TEM 36 has vonWillebrand Factor (vWF) type A domains, Fibronectin type III domains,and thrombospondin N-terminal like domain. TEM 36 is expressed inresponse to stress environment. TEM 36 may organize extracellular matrixarchitecture and be involved in matrix remodeling. There are twoisoforms of the protein, a long form and a short form. The short form ismissing amino acids 25-1188, and therefore nucleotides 73 to 3564. Bothforms share the signal sequence and are therefore both secreted.

TEM 37 is lumican, an extracellular matrix sulfated proteoglycan havinga signal sequence at residues 1-18. Lumican interacts with proteins thatare involved in matrix assembly such as collagen type I and type VI; itis involved in cell proliferation and tissue morphogenesis. Lumicanplays an important role in the regulation of collagen fiber assembly.Variations have been observed at nucleotides 1021(G,T), 1035(A,G),1209(A,G), 1259(A,C), 1418(C,A), 1519(T,A). TEM 37 is a binding partnerof TGF-β. See FASEB J. 15:559-61, 2000. One assay that can be used todetermine TEM 37 activity is a collagen fibril formation/sedimentationassay. Svensson et al., FEBS Letters 470:178-82, 2000.

TEM 38 is collagen type I, alpha 1 (COL1A1), an extracellular matrixprotein having a signal sequence at residues 1-22. Type I collagenpromotes endothelial cell migration and vascularization and induces tubeformation and is involved in tissue remodelling. Telopeptide derivativeis used as a marker for malignancy and invasion for certain cancertypes. Variations have been observed at nucleotides 296(T,G), 1810(G,A),1890(G,A), 2204(T,A), 3175(G,C), 3578(C,T), 4298(C,T), 4394(A,T),4410(A,C), 4415(C.A), 4419 (A,T), 4528(C,A), 4572(G,T), 4602(T,C),5529(T,C), 5670(C,T), 5985(C,T), 6012(C,T).

TEM 39 is transforming growth factor β-3 (TGF-beta3). It has a signalsequence at residues 1-23. It is a secreted protein. TEM 39 regulatescell growth and differentiation. TGF-beta isoforms play a major role invascular repair processes and remodeling. Variations have been observedat nucleotide 2020(G,T).

TEM 41 is similar to Olfactomedin like protein. It appears to be anintracellular protein, having no obvious predicted signal sequence.Olfactomedin is the major glycoprotein of the extracellular mucousmatrix of olfactory neuroepithelium. TEM 41 shares homology withlatrophilin (extracellular regions) which has cell-adhesive typedomains. TEM 41 may be involved in adhesive function.

TEM 42 is MSTP032 protein, a cell surface protein having a trasmembranedomain at residues 42-61. Its function is unknown and it shares littlehomology with other proteins. Variations have been observed atnucleotides 418(A,T), 724(C,A).

TEM 44 is a hypothetical protein FLJ1190 (NM_(—)018354) which has twopredicted transmembrane domains at residues 121-143 and 176-197.Residues 144-175 may form an extracellular region. TEM 44's function isnot known and shares no homology to other known proteins.

TEM 45 is tropomyosin 1 (alpha), a protein which is intracellular. Itforms dimers with a beta subunit. It influences actin function. TEM 45may be involved in endothelial cell cytoskeletal rearrangement.Variations have been observed at nucleotides 509(A,C), 621(A,C),635(T,G), 642(C,G), 1059(G,T).

TEM 46 is peanut-like 1 protein/septin 5, which belongs to the septinfamily. Proteins in the septin family bind to GTP andphosphatidylinositol 4,5-bisphosphate. They are involved in the signaltranduction cascades controlling cytokinesis and cell division.

NEM 4 is a member of the small inducible cytokine subfamily A (cys-cys),member 14 (SCYA14). NEM4 is a secreted protein characterized by twoadjacent cysteine residues. One isoform lacks internal 16 amino acidscompared to isoform 2.

NEM 22 shares homology with guanylate kinase-interacting protein1Maguin-1. It is a membrane associated protein.

NEM 23 is human signaling lymphocytic acitavation molecule (SLAM). Ithas a signal sequence at residues 1-20. The extracellular domain mayreside at residues 21-237. There is a secreted isoform of the protein.

NEM33 is netrin 4. It induces neurite outgrowth and promotes vasculardevelopment. At higher concentration, neurite outgrowth is inhibited.

ECs represent only aminor fraction of the total cells within normal ortumor tissues, and only those EC transcripts expressed at the highestlevels would be expected to be represented in libraries constructed fromunfractionated tissues. The genes described in the current study shouldtherefore provide a valuable resource for basic and clinical studies ofhuman angiogenesis in the future. Genes which have been identified astumor endothelial markers (TEMs) correspond to tags shown in SEQ ID NOS:94-139, 173-176, 180-186. Genes which have been identified as normalendothelial markers (NEMs) correspond to tags shown in SEQ ID NOS:140-172. Genes which have been identified as pan-endothelial markers(PEMs) i.e., expressed in both tumor and normal endothelial cellscorrespond to tags shown in SEQ ID NOS: 1-93. Genes which have beenpreviously identified as being expressed predominantly in the endothehumcorrespond to PEM tags shown in SEQ ID NOS: 1-6, 8, 10-15. Markers ineach class can be used interchangeably for some purposes.

Isolated and purified nucleic acids, according to the present inventionare those which are not linked to those genes to which they are linkedin the human genome. Moreover, they are not present in a mixture such asa library containing a multitude of distinct sequences from distinctgenes. They may be, however, linked to other genes such as vectorsequences or sequences of other genes to which they are not naturallyadjacent. Tags disclosed herein, because of the way that they were made,represent sequences which are 3′ of the 3′ most restriction enzymerecognition site for the tagging enzyme used to generate the SAGE tags.In this case, the tags are 3′ of the most 3′ most NlaIII site in thecDNA molecules corresponding to mRNA. Nucleic acids corresponding totags may be RNA, cDNA, or genomic DNA, for example. Such correspondingnucleic acids can be determined by comparison to sequence databases todetermine sequence identities. Sequence comparisons can be done usingany available technique, such as BLAST, available from the NationalLibrary of Medicine, National Center for Biotechnology Information. Tagscan also be used as hybridization probes to libraries of genomic or cDNAto identify the genes from which they derive. Thus, using sequencecomparisons or cloning, or combinations of these methods, one skilled inthe art can obtain full-length nucleic acid sequences. Genescorresponding to tags will contain the sequence of the tag at the 3′ endof the coding sequence or of the 3′ untranslated region (UTR), 3′ of the3′ most recognition site in the cDNA for the restriction endonucleasewhich was used to make the tags. The nucleic acids may represent eitherthe sense or the anti-sense strand. Nucleic acids and proteins althoughdisclosed herein with sequence particularity, may be derived from asingle individual. Allelic variants which occur in the population ofhumans are including within the scope of such nucleic acids andproteins. Those of skill in the art are well able to identify allelicvariants as being the same gene or protein Given a nucleic acid, one ofordinary skill in the art can readily determine an open reading framepresent, and consequently the sequence of a polypeptide encoded by theopen reading frame and, using techniques well known in the art, expresssuch protein in a suitable host. Proteins comprising such polypeptidescan be the naturally occurring proteins, fusion proteins comprisingexogenous sequences from other genes from humans or other species,epitope tagged polypeptides, etc. Isolated and purified proteins are notin a cell, and are separated from the normal cellular constituents, suchas nucleic acids, lipids, etc. Typically the protein is purified to suchan extent that it comprises the predominant species of protein in thecomposition, such as greater than 50, 60 70, 80, 90, or even 95% of theproteins present.

Using the proteins according to the invention, one of ordinary skill inthe art can readily generate antibodies which specifically bind to theproteins. Such antibodies can be monoclonal or polyclonal. They can bechimeric, humanized, or totally human. Any functional fragment orderivative of an antibody can be used including Fab, Fab′, Fab2, Fab′2,and single chain variable regions. So long as the fragment or derivativeretains specificity of binding for the endothelial marker protein it canbe used. Antibodies can be tested for specificity of binding bycomparing binding to appropriate antigen to binding to irrelevantantigen or antigen mixture under a given set of conditions. If theantibody binds to the appropriate antigen at least 2, 5, 7, andpreferably 10 times more than to irrelevant antigen or antigen mixturethen it is considered to be specific.

Techniques for making such partially to fully human antibodies are knownin the art and any such techniques can be used. According to oneparticularly preferred embodiment, fully human antibody sequences aremade in a transgenic mouse which has been engineered to express humanheavy and light chain antibody genes. Multiple strains of suchtransgenic mice have been made which can produce different classes ofantibodies. B cells from transgenic mice which are producing a desirableantibody can be fused to make hybridoma cell lines for continuousproduction of the desired antibody. See for example, Nina D. Russel,Jose R. F. Corvalan, Michael L. Gallo, C. Geoffrey Davis, Liise-AnnePirofski. Production of Protective Human Antipneumococcal Antibodies byTransgenic Mice with Human Immunoglobulin Loci Infection and ImmunityApril 2000, p. 1820-1826; Michael L. Gallo, Vladimir E. Ivanov, AyaJakobovits, and C. Geoffrey Davis. The human immunoglobulin lociintroduced into mice: V (D) and J gene segment usage similar to that ofadult humans European Journal of Immunology 30: 534-540, 2000; Larry L.Green. Antibody engineering via genetic engineering of the mouse:XenoMouse strains are a vehicle for the facile generation of therapeutichuman monoclonal antibodies Journal of Immunological Methods 231 11-23,1999; Yang X-D, Corvalan J R F, Wang P, Roy CM-N and Davis C G. FullyHuman Anti-interleukin-8 Monoclonal Antibodies: Potential Therapeuticsfor the Treatment of Inflammatory Disease States. Journal of LeukocyteBiology Vol. 66, pp 401-410 (1999); Yang X-D, Jia X-C, Corvalan J R F,Wang P, C G Davis and Jakobovits A. Eradication of Established Tumors bya Fully Human Monoclonal Antibody to the Epidermal Growth FactorReceptor without Concomitant Chemotherapy. Cancer Research Vol. 59,Number 6, pp 1236-1243 (1999); Jakobovits A. Production and selection ofantigen-specific fully human monoclonal antibodies from mice engineeredwith human Ig loci. Advanced Drug Delivery Reviews Vol. 31, pp: 33-42(1998); Green L and Jakobovits A. Regulation of B cell development byvariable gene complexity in mice reconstituted with human immunoglobulinyeast artificial chromosomes. J. Exp. Med. Vol. 188, Number 3, pp:483-495 (1998); Jakobovits A. The long-awaited magic bullets:therapeutic human monoclonal antibodies from transgenic mice. Exp. Opin.Invest. Drugs Vol. 7(4), pp: 607-614 (1998); Tsuda H, Maynard-Currie K,Reid L, Yoshida T, Edamura K, Maeda N, Smithies O. Jakobovits A.Inactivation of Mouse HPRT locus by a 203-bp retrotransposon insertionand a 55-kb gene-targeted deletion: establishment of new HPRT-Deficientmouse embryonic stem cell lines. Genomics Vol. 42, pp: 413-421 (1997);Sherman-Gold, R. Monoclonal Antibodies: The Evolution from '80s MagicBullets To Mature, Mainstream Applications as Clinical Therapeutics.Genetic Engineering News Vol. 17, Number 14 (August 1997); Mendez M,Green L, Corvalan J, Jia X-C, Maynard-Currie C, Yang X-d, Gallo M, LouieD, Lee D, Erickson K, Luna J, Roy C, Abderrahim H, Kirschenbaum F,Noguchi M, Smith D, Fukushima A, Hales J, Finer M, Davis C, Zsebo K,Jakobovits A. Functional transplant of megabase human immunoglobulinloci recapitulates human antibody response in mice. Nature Genetics Vol.15, pp: 146-156 (1997); Jakobovits A. Mice engineered with humanimmunoglobulin YACs: A new technology for production of fully humanantibodies for autoimmunity therapy. Weir's Handbook of ExperimentalImmunology, The Integrated Immune System Vol. IV, pp: 194.1-194.7(1996); Jakobovits A. Production of fully human antibodies by transgenicmice. Current Opinion in Biotechnology Vol. 6, No. 5, pp: 561-566(1995); Mendez M, Abderrahim H, Noguchi M, David N, Hardy M, Green L,Tsuda H, Yoast S, Maynard-Currie C, Garza D, Gemmill R, Jakobovits A,Klapholz S. Analysis of the structural integrity of YACs comprisinghuman immunoglobulin genes in yeast and in embryonic stem cells.Genomics Vol. 26, pp: 294-307 (1995); Jakobovits A. YAC Vectors:Humanizing the mouse genome. Current Biology Vol. 4, No. 8, pp: 761-763(1994); Arbones M, Ord D, Ley K, Ratech H, Maynard-Curry K, Otten G,Capon D, Tedder T. Lymphocyte homing and leukocyte rolling and migrationare impaired in L-selectin-deficient mice. Immunity Vol. 1, No. 4, pp:247-260 (1994); Green L, Hardy M, Maynard-Curry K, Tsuda H, Louie D,Mendez M, Abderrahim H, Noguchi M, Smith D, Zeng Y, et. al.Antigen-specific human monoclonal antibodies from mice engineered withhuman Ig heavy and light chain YACs. Nature Genetics Vol. 7, No. 1, pp:13-21 (1994); Jakobovits A, Moore A, Green L, Vergara G, Maynard-CurryK, Austin H, Klapholz S. Germ-line transmission and expression of ahuman-derived yeast artificial chromosome. Nature Vol. 362, No. 6417,pp: 255-258 (1993) Jakobovits A, Vergara G, Kennedy J, Hales J,McGuinness R, Casentini-Borocz D, Brenner D, Otten G. Analysis ofhomozygous mutant chimeric mice: deletion of the immunoglobulinheavy-chain joining region blocks B-cell development and antibodyproduction. Proceedings of the National Academy of Sciences USA Vol. 90,No. 6, pp: 2551-2555 (1993); Kucherlapati et al., U.S. Pat. No.6,1075,181.

Antibodies can also be made using phage display techniques. Suchtechniques can be used to isolate an initial antibody or to generatevariants with altered specificity or avidity characteristics. Singlechain Fv can also be used as is convenient. They can be made fromvaccinated transgenic mice, if desired. Antibodies can be produced incell culture, in phage, or in various animals, including but not limitedto cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs,cats, monkeys, chimpanzees, apes.

Antibodies can be labeled with a detectable moiety such as a radioactiveatom, a chromophore, a fluorophore, or the like. Such labeled antibodiescan be used for diagnostic techniques, either in vivo, or in an isolatedtest sample. Antibodies can also be conjugated, for example, to apharmaceutical agent, such as chemotherapeutic drug or a toxin. They canbe linked to a cytokine, to a ligand, to another antibody. Suitableagents for coupling to antibodies to achieve an anti-tumor effectinclude cytokines, such as interleukin 2 (IL-2) and Tumor NecrosisFactor (TNF); photosensitizers, for use in photodynamic therapy,including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin,and phthalocyanine; radionuclides, such as iodine-131 (¹³¹I), yttrium-90(⁹⁰Y), bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), technetium-99m(^(99m)Tc), rhenium-186 (¹⁸⁶Re), and rhenium-188 (¹⁸⁸Re); antibiotics,such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin,neocarzinostatin, and carboplatin; bacterial, plant, and other toxins,such as diphtheria toxin, pseudomonas exotoxin A, staphylococcalenterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and nativericin A), TGF-alpha toxin, cytotoxin from chinese cobra (naja najaatra), and gelonin (a plant toxin); ribosome inactivating proteins fromplants, bacteria and fungi, such as restrictocin (a ribosomeinactivating protein produced by Aspergillus restrictus), saporin (aribosome inactivating protein from Saponaria officinalis), and RNase;tyrosine kinase inhibitors; ly207702 (a difluorinated purinenucleoside); liposomes containing antitumor agents (e.g., antisenseoligonucleotides, plasmids which encode for toxins, methotrexate, etc.);and other antibodies or antibody fragments, such as F(ab).

Those of skill in the art will readily understand and be able to makesuch antibody derivatives, as they are well known in the art. Theantibodies may be cytotoxic on their own, or they may be used to delivercytotoxic agents to particular locations in the body. The antibodies canbe administered to individuals in need thereof as a form of passiveimmunization.

Characterization of extracellular regions for the cell surface andsecreted proteins from the protein sequence is based on the predictionof signal sequence, transmembrane domains and functional domains.Antibodies are preferably specifically immunoreactive with membraneassociated proteins, particularly to extracellular domains of suchproteins or to secreted proteins. Such targets are readily accessible toantibodies, which typically do not have access to the interior of cellsor nuclei. However, in some applications, antibodies directed tointracellular proteins may be useful as well. Moreover, for diagnosticpurposes, an intracellular protein may be an equally good target sincecell lysates may be used rather than a whole cell assay.

Computer programs can be used to identify extracellular domains ofproteins whose sequences are known. Such programs include SMART software(Schultz et al., Proc. Natl. Acad. Sci. USA 95: 5857-5864, 1998) andPfam software (Bateman et al., Nucleic acids Res. 28: 263-266, 2000) aswell as PSORTII. Typically such programs identify transmembrane domains;the extracellular domains are identified as immediately adjacent to thetransmembrane domains. Prediction of extracellular regions and thesignal cleavage sites are only approximate. It may have a margin oferror + or −5 residues. Signal sequence can be predicted using threedifferent methods (Nielsen et al, Protein Engineering 10: 1-6, 1997,Jagla et. al, Bioinformatics 16: 245-250, 2000, Nakai, K and Horton, P.Trends in Biochem. Sci. 24:34-35, 1999) for greater accuracy. Similarlytransmembrane (TM) domains can be identified by multiple predictionmethods. (Pasquier, et. al, Protein Eng. 12:381-385, 1999, Sonnhammer etal., In Proc. of Sixth Int. Conf. on Intelligent Systems for MolecularBiology, p. 175-182, Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop,D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998, Klein,et. al, Biochim. Biophys. Acta, 815:468, 1985, Nakai and KanehisaGenomics, 14: 897-911, 1992). In ambiguous cases, locations offunctional domains in well characterized proteins are used as a guide toassign a cellular localization.

Putative functions or functional domains of novel proteins can beinferred from homologous regions in the database identified by BLASTsearches (Altschul et. al. Nucleic Acid Res. 25: 3389-3402, 1997) and/orfrom a conserved domain database such as Pfam (Bateman et.al, NucleicAcids Res. 27:260-262 1999) BLOCKS (Henikoff, et. al, Nucl. Acids Res.28:228-230, 2000) and SMART (Ponting, et. al, Nucleic Acid Res.27,229-232, 1999). Extracellular domains include regions adjacent to atransmembrane domain in a single transmembrane domain protein (out-in ortype I class). For multiple transmembrane domains proteins, theextracellular domain also includes those regions between two adjacenttransmembrane domains (in-out and out-in). For type II transmembranedomain proteins, for which the N-terminal region is cytoplasmic, regionsfollowing the transmembrane domain is generally extracellular. Secretedproteins on the other hand do not have a transmembrane domain and hencethe whole protein is considered as extracellular.

Membrane associated proteins can be engineered to delete thetransmembrane domains, thus leaving the extracellular portions which canbind to ligands. Such soluble forms of transmembrane receptor proteinscan be used to compete with natural forms for binding to ligand. Thussuch soluble forms act as inhibitors. and can be used therapeutically asanti-angiogenic agents, as diagnostic tools for the quantification ofnatural ligands, and in assays for the identification of small moleculeswhich modulate or mimic the activity of a TEM:ligand complex.

Alternatively, the endothelial markers themselves can be used asvaccines to raise an immune response in the vaccinated animal or human.For such uses, a protein, or immunogenic fragment of such protein,corresponding to the intracellular, extracellular or secreted TEM ofinterest is administered to a subject. The immogenic agent may beprovided as a purified preparation or in an appropriately expressingcell. The administration may be direct, by the delivery of theimmunogenic agent to the subject, or indirect, through the delivery of anucleic acid encoding the immunogenic agent under conditions resultingin the expression of the immunogenic agent of interest in the subject.The TEM of interest may be delivered in an expressing cell, such as apurified population of tumor endothelial cells or a populations of fusedtumor endothelial and dendritic cells. Nucleic acids encoding the TEM ofinterest may be delivered in a viral or non-viral delivery vector orvehicle. Non-human sequences encoding the human TEM of interest or othermammalian homolog can be used to induce the desired immunologic responsein a human subject. For several of the TEMs of the present invention,mouse, rat or other ortholog sequences are described herein or can beobtained from the literature or using techniques well within the skillof the art.

Endothelial cells can be identified using the markers which aredisclosed herein as being endothelial cell specific. These include thehuman markers identified by SEQ ID NOS: 1-172, i.e., the normal,pan-endothelial, and the tumor endothelial markers. Homologous mousemarkers include tumor endothelial markers of SEQ ID NO: 182-186 and190-194. Antibodies specific for such markers can be used to identifysuch cells, by contacting the antibodies with a population of cellscontaining some endothelial cells. The presence of cross-reactivematerial with the antibodies identifies particular cells as endothelial.Similarly, lysates of cells can be tested for the presence ofcross-reactive material. Any known format or technique for detectingcross-reactive material can be used including, immunoblots,radioimmunoassay, ELISA, immunoprecipitation, and immunohistochemistry.In addition, nucleic acid probes for these markers can also be used toidentify endothelial cells. Any hybridization technique known in the artincluding Northern blotting, RT-PCR, microarray hybridization, and insitu hybridization can be used.

One can identify tumor endothelial cells for diagnostic purposes,testing cells suspected of containing one or more TEMs. One can testboth tissues and bodily fluids of a subject. For example, one can test apatient's blood for evidence of intracellular and membrane associatedTEMs, as well as for secreted TEMs. Intracellular and/or membraneassociated TEMs may be present in bodily fluids as the result of highlevels of expression of these factors and/or through lysis of cellsexpressing the TEMs.

Populations of various types of endothelial cells can also be made usingthe antibodies to endothelial markers of the invention. The antibodiescan be used to purify cell populations according to any technique knownin the art, including but not limited to fluorescence activated cellsorting. Such techniques permit the isolation of populations which areat least 50, 60, 70, 80, 90, 92, 94, 95, 96, 97, 98, and even 99% thetype of endothelial cell desired, whether normal, tumor, orpan-endothelial. Antibodies can be used to both positively select andnegatively select such populations. Preferably at least 1, 5, 10, 15,20, or 25 of the appropriate markers are expressed by the endothelialcell population.

Populations of endothelial cells made as described herein, can be usedfor screening drugs to identify those suitable for inhibiting the growthof tumors by virtue of inhibiting the growth of the tumor vasculature.

Populations of endothelial cells made as described herein, can be usedfor screening candidate drugs to identify those suitable for modulatingangiogenesis, such as for inhibiting the growth of tumors by virtue ofinhibiting the growth of endothelial cells, such as inhibiting thegrowth of the tumor or other undesired vasculature, or alternatively, topromote the growth of endothelial cells and thus stimulate the growth ofnew or additional large vessel or microvasculature.

Inhibiting the growth of endothelial cells means either regression ofvasculature which is already present, or the slowing or the absence ofthe development of new vascularization in a treated system as comparedwith a control system. By stimulating the growth of endothelial cells,one can influence development of new (neovascularization) or additionalvasculature development (revascularization). A variety of model screensystems are available in which to test the angiogenic and/oranti-angiogenic properties of a given candidate drug. Typical testsinvolve assays measuring the endothelial cell response, such asproliferation, migration, differentiation and/or intracellularinteraction of a given candidate drug. By such tests, one can study thesignals and effects of the test stimuli. Some common screens involvemeasurement of the inhibition of heparanase, endothelial tube formationon Matrigel, scratch induced motility of endothelial cells,platelet-derived growth factor driven proliferation of vascular smoothmuscle cells, and the rat aortic ring assay (which provides an advantageof capillary formation rather than just one cell type).

Drugs can be screened for the ability to mimic or modulate, inhibit orstimulate, growth of tumor endothelium cells and/or normal endothelialcells. Drugs can be screened for the ability to inhibit tumorendothelium growth but not normal endothelium growth or survival.Similarly, human cell populations, such as normal endotheliumpopulations or tumor endothelial cell populations, can be contacted withtest substances and the expression of tumor endothelial markers and/ornormal endothelial markers determined. Test substances which decreasethe expression of tumor endothelial markers (TEMs) are candidates forinhibiting angiogenesis and the growth of tumors. Conversely, markerswhich are only expressed in normal endothelium but not in tumorendothelium (NEMs) can be monitored. Test substances which increase theexpression of such NEMs in tumor endothelium and other human cells canbe identified as candidate antitumor or anti-angiogenic drugs In caseswhere the activity of a TEM or NEM is known, agents can be screened fortheir ability to decrease or increase the activity.

For those tumor endothelial markers identified as containingtransmembrane regions, it is desirable to identify drug candidatescapable of binding to the TEM receptors found at the cell surface. Forsome applications, the identification of drug candidates capable ofblocking the TEM receptor from its native ligand will be desired. Forsome applications, the identification of a drug candidate capable ofbinding to the TEM receptor may be used as a means to deliver atherapeutic or diagnostic agent. For other applications, theidentification of drug candidates capable of mimicing the activity ofthe native ligand will be desired. Thus, by manipulating the binding ofa transmembrane TEM receptor:ligand complex, one may be able to promoteor inhibit further development of endothelial cells and hence,vascularization.

For those tumor endothelial markers identified as being secretedproteins, it is desirable to identify drug candidates capable of bindingto the secreted TEM protein. For some applications, the identificationof drug candidates capable of interfering with the binding of thesecreted TEM it is native receptor. For other applications, theidentification of drug candidates capable of mimicing the activity ofthe native receptor will be desired. Thus, by manipulating the bindingof the secreted TEM:receptor complex, one may be able to promote orinhibit further development of endothelial cells, and hence,vascularization.

Expression can be monitored according to any convenient method. Proteinor mRNA can be monitored. Any technique known in the art for monitoringspecific genes' expression can be used, including but not limited toELISAs, SAGE, microarray hybridization, Western blots. Changes inexpression of a single marker may be used as a criterion for significanteffect as a potential pro-angiogenic, anti-angiogenic or anti-tumoragent. However, it also may be desirable to screen for test substanceswhich are able to modulate the expression of at least 5, 10, 15, or 20of the relevant markers, such as the tumor or normal endothelialmarkers. Inhibition of TEM protein activity can also be used as a drugscreen. Human and mouse TEMS can be used for this purpose.

Test substances for screening can come from any source. They can belibraries of natural products, combinatorial chemical libraries,biological products made by recombinant libraries, etc. The source ofthe test substances is not critical to the invention. The presentinvention provides means for screening compounds and compositions whichmay previously have been overlooked in other screening schemes. Nucleicacids and the corresponding encoded proteins of the markers of thepresent invention can be used therapeutically in a variety of modes.NEMs, can be used to restrict, diminish, reduce, or inhibitproliferation of tumor or other abnormal or undesirable vasculature.TEMs can be used to stimulate the growth of vasculature, such as forwound healing or to circumvent a blocked vessel. The nucleic acids andencoded proteins can be administered by any means known in the art. Suchmethods include, using liposomes, nanospheres, viral vectors, non-viralvectors comprising polycations, etc. Suitable viral vectors includeadenovirus, retroviruses, and sindbis virus. Administration modes can beany known in the art, including parenteral, intravenous, intramuscular,intraperitoneal, topical, intranasal, intrarectal, intrabronchial, etc.

Specific biological antagonists of TEMs can also be used to therapeuticbenefit. For example, antibodies, T cells specific for a TEM, antisenseto a TEM, and ribozymes specific for a TEM can be used to restrict,inhibit, reduce, and/or diminish tumor or other abnormal or undesirablevasculature growth. Such antagonists can be administered as is known inthe art for these classes of antagonists generally. Anti-angiogenicdrugs and agents can be used to inhibit tumor growth, as well as totreat diabetic retinopathy, rheumatoid arthritis, psoriasis, polycystickidney disease (PKD), and other diseases requiring angiogenesis fortheir pathologies.

Mouse counterparts to human TEMS can be used in mouse cancer models orin cell lines or in vitro to evaluate potential anti-angiogenic oranti-tumor compounds or therapies. Their expression can be monitored asan indication of effect. Mouse TEMs are disclosed in SEQ ID NO: 182-186and 190-194. Mouse TEMs can be used as antigens for raising antibodieswhich can be tested in mouse tumor models. Mouse TEMs with transmembranedomains are particularly preferred for this purpose. Mouse TEMs can alsobe used as vaccines to raise an immunological response in a human to thehuman ortholog.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1

Visualization of Vasculature of Colorectal Cancers

The endothelium of human colorectal cancer was chosen to address theissues of tumor angiogenesis, based on the high incidence, relativelyslow growth, and resistance to anti-neoplastic agents of these cancers.While certain less common tumor types, such as glioblastomas, are highlyvascularized and are regarded as good targets for anti-angiogenictherapy, the importance of angiogenesis for the growth of humancolorectal cancers and other common solid tumor types is less welldocumented.

We began by staining vessels in colorectal cancers using von WillebrandFactor (vWF) as a marker. In each of 6 colorectal tumors, thisexamination revealed a high density of vessels throughout the tumorparenchyma (Examples in FIGS. 1A and B). Interestingly, these analysesalso substantiated the importance of these vessels for tumor growth, asendothelium was often surrounded by a perivascular cuff of viable cells,with a ring of necrotic cells evident at the periphery (Example in FIG.1A). Although these preliminary studies suggested that colon tumors areangiogenesis-dependent, reliable markers that could distinguish vesselsin colon cancers from the vessels in normal colon are currently lacking.One way to determine if such markers exist is by analyzing geneexpression profiles in endothelium derived from normal and neoplastictissue.

EXAMPLE 2

Purification of Endothelial Cells

Global systematic analysis of gene expression in tumor and normalendothelium has been hampered by at least three experimental obstacles.First, endothelium is enmeshed in a complex tissue consisting of vesselwall components, stromal cells, and neoplastic cells, requiring highlyselective means of purifying ECs for analysis. Second, techniques fordefining global gene expression profiles were not available untilrecently. And third, only a small fraction of the cells within a tumorare endothelial, mandating the development of methods that are suitablefor the analysis of global expression profiles from relatively fewcells.

To overcome the first obstacle, we initially attempted to purify ECsfrom dispersed human colorectal tissue using CD31, an endothelial markercommonly used for this purpose. This resulted in a substantialenrichment of ECs but also resulted in contamination of the preparationsby hematopoietic cells, most likely due to expression of CD31 bymacrophages. We therefore developed a new method for purifying ECs fromhuman tissues using P1H12, a recently described marker for ECs. UnlikeCD31, P1H12 was specifically expressed on the ECs of both colorectaltumors and normal colorectal mucosa. Moreover, immunofluorescencestaining of normal and cancerous colon with a panel of known cellsurface endothelial markers (e.g. VE-cadherin, CD31 and CD34) revealedthat P1H12 was unique in that it stained all vessels includingmicrovessels (see FIG. 2A and data not shown). In addition to selectionwith P1H 12, it was necessary to optimize the detachment of ECs fromtheir neighbors without destroying their cell surface proteins as wellas to employ positive and negative affinity purifications using acocktail of antibodies (FIG. 2B). The ECs purified from normalcolorectal mucosa and colorectal cancers were essentially free ofepithelial and hematopoietic cells as judged by RT-PCR (FIG. 2C) andsubsequent gene expression analysis (see below).

EXAMPLE 3

Comparison of Tumor and Normal Endothelial Cell Expression Patterns

To overcome the remaining obstacles, a modification of the SerialAnalysis of Gene Expression (SAGE) technique was used. SAGE associatesindividual mRNA transcripts with 14 base pair tags derived from aspecific position near their 3′ termini. The abundance of each tagprovides a quantitative measure of the transcript level present withinthe mRNA population studied. SAGE is not dependent on pre-existingdatabases of expressed genes, and therefore provides an unbiased view ofgene expression profiles. This feature is particularly important in theanalysis of cells that constitute only a small fraction of the tissueunder study, as transcripts from these cells are unlikely to be wellrepresented in extant EST databases. We adapted the SAGE protocol sothat it could be used on small numbers of purified ECs obtained from theprocedure outlined in FIG. 2B. A library of ˜100,000 tags from thepurified ECs of a colorectal cancer, and a similar library from the ECsof normal colonic mucosa from the same patient were generated. These˜193,000 tags corresponded to over 32,500 unique transcripts.Examination of the expression pattern of hematopoietic, epithelial andendothelial markers confirmed the purity of the preparations (FIG. 2D).

EXAMPLE 4

Markers of Normal and Tumor Endothelium

We next sought to identify Pan Endothelial Markers (PEMs), that is,transcripts that were expressed at significantly higher levels in bothnormal and tumor associated endothelium compared to other tissues. Toidentify such PEMs, tags expressed at similar levels in both tumor andnormal ECs were compared to ˜1.8 million tags from a variety of celllines derived from tumors of non-endothelial origin. This simplecomparison identified 93 transcripts that were strikingly EC-specific,i.e. expressed at levels at least 20-fold higher in ECs in vivo comparedto non-endothelial cells in culture. The 15 tags corresponding tocharacterized genes which were most highly and specifically expressed inendothelium are shown in Table 1A. Twelve of these 15 most abundantendothelial transcripts had been previously shown to be preferentiallyexpressed in endothelium, while the other 3 genes had not beenassociated with endothelium in the past (Table 1A). These data sets alsorevealed many novel PEMs, which became increasingly prevalent as tagexpression levels decreased (Table 1B). For many of the transcripts,their endothelial origin was confirmed by SAGE analysis of 401,000transcripts derived from primary cultures of human umbilical veinendothelial cells (HUVEC) and human dermal microvascular endothelialcells (HMVEC) (Table 1A and B). To further validate the expression ofthese PEMs in vivo, we developed a highly sensitive non-radioactive insitu hybridization method that allowed the detection of transcriptsexpressed at relatively low levels in frozen sections of human tissues.Two uncharacterized markers, PEM3 and PEM6, were chosen for thisanalysis. In each case, highly specific expression was clearly limitedto vascular ECs in both normal and neoplastic tissues (FIGS. 3A and Band data not shown). These data also suggest that ECs maintained inculture do not completely recapitulate expression patterns observed invivo. For example, Hevin and several other PEM's were expressed at highlevels in both tumor and normal ECs in vivo, but few or no transcriptswere detected in cultured HUVEC or HMVEC (Table 1). The source of theHevin transcripts was confirmed to be endothelium by in situhybridization in normal and malignant colorectal tissue (FIG. 3C).

Many of the markers reported in Table 1 were expressed at significantlyhigher levels than previously characterized genes commonly associatedwith ECs. For example, the top 25 markers were all expressed at greaterthan 200 copies per cell. In contrast, the receptors for VEGF (VEGFR-1and VEGFR-2) were expressed at less than 20 copies per cell.Interestingly, VEGFR2 (KDR), which had previously been reported to beup-regulated in vessels during colon cancer progression, was found to beexpressed in both normal and neoplastic colorectal tissue (FIGS. 3D andE). The lack of specificity of this gene was in accord with the SAGEdata, which indicated that the VEGFR was expressed at 12 copies per cellin both normal and tumor endothelium.

EXAMPLE 5

Tumor Versus Normal Endothelium

We next attempted to identify transcripts that were differentiallyexpressed in endothelium derived from normal or neoplastic tissues. Thiscomparison revealed 33 tags that were preferentially expressed innormal-derived endothelium at levels at least 10-fold higher than intumor-derived endothelium. Conversely, 46 tags were expressed at 10-foldor higher levels in tumor vessels. Because those transcripts expressedat higher levels in tumor endothelium are most likely to be useful inthe future for diagnostic and therapeutic purposes, our subsequentstudies focussed on this class. Of the top 25 tags most differentiallyexpressed, 12 tags corresponded to 11 previously identified genes, onewith an alternative polyadenylation site (see Table 2). Of these 10genes, 6 have been recognized as markers associated with angiogenicvessels. The remaining 14 tags corresponded to uncharacterised genes,most of which have only been deposited as ESTs (Table 2).

To validate the expression patterns of these genes, we chose to focus on9 Tumor Endothelial Markers (BSC-TEM 1-9; TEM 1, 2, 5, 9, 16, 17, 19,and 22) for which EST sequences but no other information was available(Table 2). These tags were chosen simply because they were among themost differentially expressed on the list and because we were able toobtain suitable probes. In many cases, this required obtaining nearfull-length sequences through multiple rounds of sequencing and cDNAwalking (See accession numbers in Table 2). RT-PCR analysis was thenused to evaluate the expression of the corresponding transcripts inpurified ECs derived from normal and tumor tissues of two patientsdifferent from the one used to construct the SAGE libraries. As shown inFIG. 4A, the vWF gene, expected to be expressed in both normal and tumorendothelium on the basis of the SAGE data as well as previous studies,was expressed at similar levels in normal and tumor ECs from bothpatients, but was not expressed in purified tumor epithelial cells. Asexpected, PEM2 displayed a pattern similar to vWF. In contrast, all 9TEMs chosen for this analysis were prominently expressed in tumor ECs,but were absent or barely detectable in normal ECs (Table 3 and examplesin FIG. 4A). It is important to note that these RT-PCR assays wereextremely sensitive indicators of expression, and the absence ofdetectable transcripts in the normal endothelium, combined with theirpresence in tumor endothelial RNAs even when diluted 100-fold, providescompelling confirmatory evidence for their differential expression.These results also show that these transcripts were not simply expresseddifferentially in the ECs of the original patient, but werecharacteristic of colorectal cancer endothelium in general.

It could be argued that the results noted above were compromised by thepossibility that a small number of non-endothelial cells contaminatedthe cell populations used for SAGE and RT-PCR analyses, and that thesenon-endothelial cells were responsible for the striking differences inexpression of the noted transcripts. To exclude this possibility, weperformed in situ hybridization on normal and neoplastic colon tissue.In every case where transcripts could be detected (BSC-TEM 1, 3, 4, 5,7, 8, and 9; TEM 1, 5, 9, 17, and 19), they were specifically localizedto ECs (Table 3 and examples in FIGS. 4B and C). Although caution mustbe used when interpreting negative in situ hybridization results, noneof the TEMs were expressed in vascular ECs associated with normalcolorectal tissue even though vWF and Hevin were clearly expressed(Table 3).

EXAMPLE 6

Tumor Endothelium Markers are Expressed in Multiple Tumor Types

Were these transcripts specifically expressed in the endothelium withinprimary colorectal cancers, or were they characteristic of tumorendothelium in general? To address this question, we studied theexpression of a representative TEM (BSC-TEM7; TEM 17) in a livermetastasis from a colorectal cancer, a sarcoma, and in primary cancersof the lung, pancreas, breast and brain. As shown in FIG. 4, thetranscript was found to be expressed specifically in the endothelium ofeach of these cancers, whether metastatic (FIG. 4D) or primary (FIG.4E-I). Analysis of the other six TEMs, (BSC-TEM 1, 3, 4, 5, 7, 8 and 9;TEM 1, 5, 9, 17, and 19) revealed a similar pattern in lung tumors,brain tumors, and metastatic lesions of the liver (see Table 3).

EXAMPLE 7

Tumor Endothelium Markers are Neo-angiogenic

Finally, we asked whether these transcripts were expressed in angiogenicstates other than that associated with tumorigenesis. We thus performedin situ hybridizations on corpus luteum tissue as well as healingwounds. Although there were exceptions, we found that these transcriptswere generally expressed both in the corpus luteum and in thegranulation tissue of healing wounds (Table 3 and example in FIG. 4J).In all tissues studied, expression of the genes was either absent orexclusively confined to the EC compartment.

REFERENCES AND NOTES

The disclosure of each reference cited is expressly incorporated herein.

-   1. J. Folkman, in Cancer Medicine J. Holland, Bast Jr, R C, Morton D    L, Frei III, E, Kufe, D W, Weichselbaum, R R, Ed. (Williams &    Wilkins, Baltimore, 1997) pp. 181.-   2. R. S. Kerbel, Carcinogenesis 21, 505 (2000).-   3. P. Wesseling, D. J. Ruiter, P. C. Burger, J Neurooncol 32, 253    (1997).-   4. Q. G. Dong, et al., Arterioscler Thromb Vasc Biol 17, 1599    (1997).-   5. P. W. Hewett, J. C. Murray, In Vitro Cell Dev Biol Anim 32, 462    (1996).-   6. M. A. Hull, P. W. Hewett, J. L. Brough, C. J. Hawkey,    Gastroenterology 111, 1230 (1996).-   7. G. Haraldsen, et al., Gut 37, 225 (1995).-   8. The original EC isolation protocol was the same as that shown in    FIG. 2B except that dispersed cells were stained with anti-CD31    antibodies instead of anti-P1H12, and magnetic beads against CD64    and CD14 were not included in the negative selection. After    generating 120,000 SAGE tags from these two EC preparations, careful    analysis of the SAGE data revealed that, in addition to    endothelial-specific markers, several macrophage-specific markers    were also present.-   9. A. Solovey, et al., N Engl J Med 337, 1584 (1997).-   10. V. E. Velculescu, L. Zhang, B. Vogelstein, K. W. Kinzler,    Science 270, 484-487 (1995).-   11. In order to reduce the minimum amount of starting material    required from ˜50 million cells to ˜50,000 cells (i.e. ˜1000-fold    less) we and others (38) have introduced several modifications to    the original SAGE protocol. A detailed version of our modified    “MicroSAGE” protocol is available from the authors upon request.-   12. 96,694 and 96,588 SAGE tags were analyzed from normal and tumor    derived ECs, respectively, and represented 50,298 unique tags. A    conservative estimate of 32,703 unique transcripts was derived by    considering only those tags observed more than once in the current    data set or in the 134,000 transcripts previously identified in    human transcriptomes (39).-   13. To identify endothelial specific transcripts, we normalized the    number of tags analyzed in each group to 100,000, and limited our    analysis to transcripts that were expressed at levels at least    20-fold higher in ECs than in non-endothelial cell lines in culture    and present at fewer than 5 copies per 100,000 transcripts in    non-endothelial cell lines and the hematopoietic fraction (˜57,000    tags) (41). Non-endothelial cell lines consisted of 1.8×106 tags    derived from a total of 14 different cancer cell lines including    colon, breast, lung, and pancreatic cancers, as well as one    non-transformed keratinocyte cell line, two kidney epithelial cell    lines, and normal monocytes. A complete list of PEMs is available at    www.sagenet.org\angio\table1.htm.-   14. M. Tucci, et al., J Endocrinol 157, 13 (1998).-   15. T. Oono, et al., J Invest Dermatol 100, 329 (1993).-   16. K. Motamed, Int J Biochem Cell Biol 31, 1363 (1999).-   17. N. Bardin, et al., Tissue Antigens 48, 531 (1996).-   18. D. M. Bradham, A. Igarashi, R. L. Potter, G. R. Grotendorst, J    Cell Biol 114, 1285 (1991).-   19. K. Akaogi, et al., Proc Natl Acad Sci USA 93, 8384 (1996).-   20. Y. Muragaki, et al., Proc Natl Acad Sci USA 92, 8763 (1995).-   21. M. L. Iruela-Arispe, C. A. Diglio, E. H. Sage, Arterioscler    Thromb 11, 805 (1991).-   22. J. P. Girard, T. A. Springer, Immunity 2, 113 (1995).-   23. E. A. Jaffe, et al., J Immunol 143, 3961 (1989).-   24. J. P. Girard, et al., Am J Pathol 155, 2043 (1999).-   25. H. Ohtani, N. Sasano, J Electron Microsc 36, 204 (1987).-   26. For non-radioactive in situ hybridization, digoxigenin    (DIG)-labelled sense and anti-sense riboprobes were generated    through PCR by amplifying 500-600 bp products and incorporating a T7    promoter into the anti-sense primer. In vitro transcription was    performed using DIG RNA labelling reagents and T7 RNA polymerase    (Roche, Indianapolis, Ind.). Frozen tissue sections were fixed with    4% paraformaldehyde, penneabilized with pepsin, and incubated with    200 ng/ml of riboprobe overnight at 55° C. For signal amplification,    a horseradish peroxidase (HRP) rabbit anti-DIG antibody (DAKO,    Carpinteria, Calif.) was used to catalyse the deposition of    Biotin-Tyramide (from GenPoint kit, DAKO). Further amplification Was    achieved by adding HRP rabbit anti-biotin (DAKO), biotin-tyramide,    and then alkaline-phosphatase (AP) rabbit anti-biotin (DAKO). Signal    was detected using the AP substrate Fast Red TR/Napthol AS-MX    (Sigma, St. Louis, Mo.), and cells were counterstained with    hematoxylin unless otherwise indicated. A detailed protocol    including the list of primers used to generate the probes can be    obtained from the authors upon request.-   27. Transcript copies per cell were calculated assuming an average    cell contains 300,000 transcripts.-   28. R. S. Warren, H. Yuan, M. R. Matli, N. A. Gillett, N. Ferrara, J    Clin Invest 95, 1789 (1995).-   29. Y. Takahashi, Y. Kitadai, C. D. Bucana, K. R. Cleary, L. M.    Ellis, Cancer Res 55, 3964 (1995).-   30. L. F. Brown, et al., Cancer Res 53, 4727 (1993).-   31. Endothelial-specific transcripts were defined as those expressed    at levels at least 5-fold higher in ECs in vivo than in    non-endothelial cell lines in culture (13), and present at no more    than 5 copies per 100,000 transcripts in non-endothelial cell lines    and the hematopoietic cell fraction (41). Transcripts showing    statistically different levels of expression (P<0.05) were then    identified using Monte Carlo analysis as previously described (40).    Transcripts preferentially expressed in normal endothelium were then    defined as those expressed at levels at least 10-fold higher in    normal endothelium than in tumor endothelium. Conversely, tumor    endothelial transcripts were at least 10-fold higher in tumor versus    normal endothelium. See www.sagenet.org\angio\table2.htm and    www.sagenet.org\angio\table3.htm for a complete list of    differentially expressed genes.-   32. M. Iurlaro, et al., Eur J Clin Invest 29, 793 (1999).-   33. W. S. Lee, et al., Circ Res 82, 845 (1998).-   34. J. Niquet, A. Represa, Brain Res Dev Brain Res 95, 227 (1996).-   35. L. Fouser, L. Iruela-Arispe, P. Bornstein, E. H. Sage, J Biol    Chem 266, 18345 (1991).-   36. M. L. Iruela-Arispe, P. Hasselaar, H. Sage, Lab Invest 64, 174    (1991).-   37. H. F. Dvorak, N Engl J Med 315, 1650 (1986).-   38. B. Virlon, et al., Proc Natl Acad Sci USA 96, 15286 (1999).-   39. V. E. Velculescu, et al., Nat Genet 23, 387 (1999).-   40. L. Zhang, et al., Science 276, 1268 (1997).-   41. Human colon tissues were obtained within ½ hour after surgical    removal from patients. Sheets of epithelial cells were peeled away    from normal tissues with a glass slide following treatment with 5 mM    DDT, then 10 mM EDTA, leaving the lamina propria intact. After a 2 h    incubation in collagenase at 37° C., cells were filtered    sequentially through 400 um, 100 um, 50 um and 25 um mesh, and spun    through a 30% pre-formed Percoll gradient to pellet RBCs. Epithelial    cells (Epithelial Fraction), which were found to non-specifically    bind magnetic beads, were removed using Dynabeads coupled to BerEP4    (Dynal, Lake Success, N.Y.). Subsequently, macrophages and other    leukocytes (Hematopoietic Fraction) were removed using a cocktail of    beads coupled to anti-CD45, anti-CD 14 and anti-CD64 (Dynal). The    remaining cells were stained with P1H12 antibody, purified with    anti-mouse IgG-coupled magnetic beads, and lysed in mRNA lysis    buffer. A detailed protocol can be obtained from the authors upon    request.-   42. H. Sheikh, H. Yarwood, A. Ashworth, C. M. Isacke, J Cell Sci    113, 1021-32 (2000).

SEQ ID SEQ ID Sequence name NO: NO: Sequence name PEM 1 1 1 PEM 1 PEM 22 2 PEM 2 PEM 3 3 3 PEM 3 PEM 4 4 4 PEM 4 PEM 5 5 5 PEM 5 PEM 6 6 6 PEM6 PEM 7 7 7 PEM 7 PEM 8 8 8 PEM 8 PEM 9 9 9 PEM 9 PEM 10 10 10 PEM 10PEM 11 11 11 PEM 11 PEM 12 12 12 PEM 12 PEM 13 13 13 PEM 13 PEM 14 14 14PEM 14 PEM 15 15 15 PEM 15 PEM 16 16 16 PEM 16 PEM 17 17 17 PEM 17 PEM18 18 18 PEM 18 PEM 19 19 19 PEM 19 PEM 20 20 20 PEM 20 PEM 21 21 21 PEM21 PEM 22 22 22 PEM 22 PEM 23 23 23 PEM 23 PEM 24 24 24 PEM 24 PEM 25 2525 PEM 25 PEM 26 26 26 PEM 26 PEM 27 27 27 PEM 27 PEM 28 28 28 PEM 28PEM 29 29 29 PEM 29 PEM 30 30 30 PEM 30 PEM 31 31 31 PEM 31 PEM 32 32 32PEM 32 PEM 33 33 33 PEM 33 PEM 34 34 34 PEM 34 PEM 35 35 35 PEM 35 PEM36 36 36 PEM 36 PEM 37 37 37 PEM 37 PEM 38 38 38 PEM 38 PEM 39 39 39 PEM39 PEM 40 40 40 PEM 40 PEM 41 41 41 PEM 41 PEM 42 42 42 PEM 42 PEM 43 4343 PEM 43 PEM 44 44 44 PEM 44 PEM 45 45 45 PEM 45 PEM 46 46 46 PEM 46PEM 47 47 47 PEM 47 PEM 48 48 48 PEM 48 PEM 49 49 49 PEM 49 PEM 50 50 50PEM 50 PEM 51 51 51 PEM 51 PEM 52 52 52 PEM 52 PEM 53 53 53 PEM 53 PEM54 54 54 PEM 54 PEM 55 55 55 PEM 55 PEM 56 56 56 PEM 56 PEM 57 57 57 PEM57 PEM 58 58 58 PEM 58 PEM 59 59 59 PEM 59 PEM 60 60 60 PEM 60 PEM 61 6161 PEM 61 PEM 62 62 62 PEM 62 PEM 63 63 63 PEM 63 PEM 64 64 64 PEM 64PEM 65 65 65 PEM 65 PEM 66 66 66 PEM 66 PEM 67 67 67 PEM 67 PEM 68 68 68PEM 68 PEM 69 69 69 PEM 69 PEM 70 70 70 PEM 70 PEM 71 71 71 PEM 71 PEM72 72 72 PEM 72 PEM 73 73 73 PEM 73 PEM 74 74 74 PEM 74 PEM 75 75 75 PEM75 PEM 76 76 76 PEM 76 PEM 77 77 77 PEM 77 PEM 78 78 78 PEM 78 PEM 79 7979 PEM 79 PEM 80 80 80 PEM 80 PEM 81 81 81 PEM 81 PEM 82 82 82 PEM 82PEM 83 83 83 PEM 83 PEM 84 84 84 PEM 84 PEM 85 85 85 PEM 85 PEM 86 86 86PEM 86 PEM 87 87 87 PEM 87 PEM 88 88 88 PEM 88 PEM 89 89 89 PEM 89 PEM90 90 90 PEM 90 PEM 91 91 91 PEM 91 PEM 92 92 92 PEM 92 PEM 93 93 93 PEM93 TEM 1 94 94 TEM 1 TEM 2 95 95 TEM 2 TEM 3 96 96 TEM 3 TEM 4 97 97 TEM4 TEM 5 98 98 TEM 5 TEM 6 99 99 TEM 6 TEM 7 100 100 TEM 7 TEM 8 101 101TEM 8 TEM 9 102 102 TEM 9 TEM 10 103 103 TEM 10 TEM 11 104 104 TEM 11TEM 12 105 105 TEM 12 TEM 13 106 106 TEM 13 TEM 14 107 107 TEM 14 TEM 15108 108 TEM 15 TEM 16 109 109 TEM 16 TEM 17 110 110 TEM 17 TEM 18 111111 TEM 18 TEM 19 112 112 TEM 19 TEM 20 113 113 TEM 20 TEM 21 114 114TEM 21 TEM 22 115 115 TEM 22 TEM 23 116 116 TEM 23 TEM 24 117 117 TEM 24TEM 25 118 118 TEM 25 TEM 26 119 119 TEM 26 TEM 27 120 120 TEM 27 TEM 28121 121 TEM 28 TEM 29 122 122 TEM 29 TEM 30 123 123 TEM 30 TEM 31 124124 TEM 31 TEM 32 125 125 TEM 32 TEM 33 126 126 TEM 33 TEM 34 127 127TEM 34 TEM 35 128 128 TEM 35 TEM 36 129 129 TEM 36 TEM 37 130 130 TEM 37TEM 38 131 131 TEM 38 TEM 39 132 132 TEM 39 TEM 40 133 133 TEM 40 TEM 41134 134 TEM 41 TEM 42 135 135 TEM 42 TEM 43 136 136 TEM 43 TEM 44 137137 TEM 44 TEM 45 138 138 TEM 45 TEM 46 139 139 TEM 46 NEM 1 140 140 NEM1 NEM 2 141 141 NEM 2 NEM 3 142 142 NEM 3 NEM 4 143 143 NEM 4 NEM 5 144144 NEM 5 NEM 6 145 145 NEM 6 NEM 7 146 146 NEM 7 NEM 8 147 147 NEM 8NEM 9 148 148 NEM 9 NEM 10 149 149 NEM 10 NEM 11 150 150 NEM 11 NEM 12151 151 NEM 12 NEM 13 152 152 NEM 13 NEM 14 153 153 NEM 14 NEM 15 154154 NEM 15 NEM 16 155 155 NEM 16 NEM 17 156 156 NEM 17 NEM 18 157 157NEM 18 NEM 19 158 158 NEM 19 NEM 20 159 159 NEM 20 NEM 21 160 160 NEM 21NEM 22 161 161 NEM 22 NEM 23 162 162 NEM 23 NEM 24 163 163 NEM 24 NEM 25164 164 NEM 25 NEM 26 165 165 NEM 26 NEM 27 166 166 NEM 27 NEM 28 167167 NEM 28 NEM 29 168 168 NEM 29 NEM 30 169 169 NEM 30 NEM 31 170 170NEM 31 NEM 32 171 171 NEM 32 NEM 33 172 172 NEM 33 TEM 1 DNA 173 173 TEM1 DNA TEM 2 DNA 174 174 TEM 2 DNA TEM 7 DNA 175 175 TEM 7 DNA TEM 8 DNA176 176 TEM 8 DNA TEM 1 Protein 177 177 TEM 1 Protein TEM 2 Protein 178178 TEM 2 Protein TEM 8 Protein 179 179 TEM 8 Protein TEM 5 DNA 180 180TEM 5 DNA TEM 7B DNA 181 181 TEM 7B DNA mTEM 1 DNA 182 182 mTEM 1 DNAmTEM 5 DNA 183 183 mTEM 5 DNA mTEM 7 DNA 184 184 mTEM 7 DNA mTEM 7B DNA185 185 mTEM 7B DNA mTEM 8 DNA 186 186 mTEM 8 DNA TEM 8 Protein 187 187TEM 8 Protein TEM 5 Protein 188 188 TEM 5 Protein TEM 7B Protein 189 189TEM 7B Protein mTEM 1 Protein 190 190 mTEM 1 Protein mTEM 5 Protein 191191 mTEM 5 Protein mTEM 7 Protein 192 192 mTEM 7 Protein mTEM 7b Protein193 193 mTEM 7b Protein mTEM 8 Protein 194 194 mTEM 8 Protein TEM 1 DNA195 195 TEM 1 DNA TEM 1 Protein 196 196 TEM 1 Protein TEM 2 DNA 197 197TEM 2 DNA TEM 2 Protein 198 198 TEM 2 Protein TEM 3 DNA 199 199 TEM 3DNA TEM 3 Protein 200 200 TEM 3 Protein TEM 4 DNA 201 201 TEM 4 DNA TEM4 Protein 202 202 TEM 4 Protein TEM 5 DNA 203 203 TEM 5 DNA TEM 5Protein 204 204 TEM 5 Protein TEM 6 DNA 205 205 TEM 6 DNA TEM 6 Protein206 206 TEM 6 Protein TEM 7 DNA 207 207 TEM 7 DNA TEM 7 Protein 208 208TEM 7 Protein TEM 8 DNA 209 209 TEM 8 DNA TEM 8 Protein 210 210 TEM 8Protein TEM 9 DNA 211 211 TEM 9 DNA TEM 9 Protein 212 212 TEM 9 ProteinTEM 10 DNA 213 213 TEM 10 DNA TEM 10 Protein 214 214 TEM 10 Protein TEM11 DNA 215 215 TEM 11 DNA TEM 11 Protein 216 216 TEM 11 Protein TEM 12DNA 217 217 TEM 12 DNA TEM 12 Protein 218 218 TEM 12 Protein TEM 13 DNA219 219 TEM 13 DNA TEM 13 Protein 220 220 TEM 13 Protein TEM 14a DNA 221221 TEM 14a DNA TEM 14b DNA 222 222 TEM 14b DNA TEM 14a Protein 223 223TEM 14a Protein TEM 14b Protein 224 224 TEM 14b Protein TEM 15 DNA 225225 TEM 15 DNA TEM 15 Protein 226 226 TEM 15 Protein TEM 16 DNA 227 227TEM 16 DNA TEM 16 Protein 228 228 TEM 16 Protein TEM 17 DNA 229 229 TEM17 DNA TEM 17 Protein 230 230 TEM 17 Protein TEM 19 DNA 231 231 TEM 19DNA TEM 19 Protein 232 232 TEM 19 Protein TEM 20 DNA 233 233 TEM 20 DNATEM 20 Protein 234 234 TEM 20 Protein TEM 21 DNA 235 235 TEM 21 DNA TEM21 Protein 236 236 TEM 21 Protein TEM 22 DNA 237 237 TEM 22 DNA TEM 22Protein 238 238 TEM 22 Protein TEM 24 DNA 239 239 TEM 24 DNA TEM 24Protein 240 240 TEM 24 Protein TEM 25 DNA 241 241 TEM 25 DNA TEM 25Protein 242 242 TEM 25 Protein TEM 27 DNA 243 243 TEM 27 DNA TEM 27Protein 244 244 TEM 27 Protein TEM 28 DNA 245 245 TEM 28 DNA TEM 28Protein 246 246 TEM 28 Protein TEM 29 DNA 247 247 TEM 29 DNA TEM 29Protein 248 248 TEM 29 Protein TEM 30 DNA 249 249 TEM 30 DNA TEM 30Protein 250 250 TEM 30 Protein TEM 31 DNA 251 251 TEM 31 DNA TEM 31Protein 252 252 TEM 31 Protein TEM 33 DNA 253 253 TEM 33 DNA TEM 33Protein 254 254 TEM 33 Protein TEM 35 DNA 255 255 TEM 35 DNA TEM 35Protein 358 256 TEM 36 DNA TEM 36 DNA 256 257 TEM 36 Protein TEM 36Protein 257 258 TEM 37 DNA TEM 37 DNA 258 259 TEM 37 Protein TEM 37Protein 259 260 TEM 38 DNA TEM 38 DNA 260 261 TEM 38 Protein TEM 38Protein 261 262 TEM 39 DNA TEM 39 DNA 262 263 TEM 39 Protein TEM 39Protein 263 264 TEM 40 DNA TEM 40 DNA 264 265 TEM 40 Protein TEM 40Protein 265 266 TEM 41 DNA TEM 41 DNA 266 267 TEM 41 Protein TEM 41Protein 267 268 TEM 42 DNA TEM 42 DNA 268 269 TEM 42 Protein TEM 42Protein 269 270 TEM 44 DNA TEM 44 DNA 270 271 TEM 44 Protein TEM 44Protein 271 272 TEM 45 DNA TEM 45 DNA 272 273 TEM 45 Protein TEM 45Protein 273 274 TEM 46 DNA TEM 46 DNA 274 275 TEM 46 Protein TEM 46Protein 275 276 NEM 4 DNA NEM 4 DNA 276 277 NEM 4 Protein NEM 4 Protein277 278 NEM 14 DNA NEM 14 DNA 278 279 NEM 14 Protein NEM 14 Protein 279280 NEM 17 DNA NEM 17 DNA 280 281 NEM 17 Protein NEM 17 Protein 281 282NEM 22 DNA NEM 22 DNA 282 283 NEM 22 Protein NEM 22 Protein 283 284 NEM23 DNA NEM 23 DNA 284 285 NEM 23 Protein NEM 23 Protein 285 286 NEM 23Secreted NEM 23 Secreted 286 287 NEM 23 Short NEM 23 Short 287 288 NEM33 DNA NEM 33 DNA 288 289 NEM 33 Protein NEM 33 Protein 289 290 mTEM 1DNA mTEM 1 DNA 290 291 mTEM 1 Protein mTEM 1 Protein 291 292 mTEM 2 DNAmTEM 2 DNA 292 293 mTEM 2 Protein mTEM 2 Protein 293 294 mTEM 9 DNA mTEM3 DNA 298 295 mTEM 9 Protein mTEM 3 Protein 299 296 mTEM 17 DNA mTEM 9DNA 294 297 mTEM 17 Protein mTEM 9 Protein 295 298 mTEM 3 DNA mTEM 13DNA 302 299 mTEM 3 Protein mTEM 13 Protein 303 300 mTEM 19 DNA mTEM 17DNA 296 301 mTEM 19 Protein mTEM 17 Protein 297 302 mTEM 13 DNA mTEM 19DNA 300 303 mTEM 13 Protein mTEM 19 Protein 301 304 mTEM 22 DNA mTEM 22DNA 304 305 mTEM 22 Protein mTEM 22 Protein 305 306 mTEM 30 DNA mTEM 30DNA 306 307 mTEM 30 Protein mTEM 30 Protein 307 308 TEM 2 tag TEM 2 tag308 309 TEM 1 long tag TEM 1 long tag 309 310 TEM 3 long tag TEM 3 longtag 310 311 TEM 4 long tag TEM 4 long tag 311 312 TEM 5 long tag TEM 5long tag 312 313 TEM 5 long tag TEM 5 long tag 313 314 TEM 6 long tagTEM 6 long tag 314 315 TEM 7 long tag TEM 7 long tag 315 316 TEM 8 longtag TEM 8 long tag 316 317 TEM 9 long tag TEM 9 long tag 317 318 TEM 10long tag TEM 10 long tag 318 319 TEM 10 long tag TEM 10 long tag 319 320TEM 10 long tag TEM 10 long tag 320 321 TEM 11 long tag TEM 11 long tag321 322 TEM 12 long tag TEM 12 long tag 322 323 TEM 13 long tag TEM 13long tag 323 324 TEM 13 long tag TEM 13 long tag 324 325 TEM 14 long tagTEM 14 long tag 325 326 TEM 15 long tag TEM 15 long tag 326 327 TEM 15long tag TEM 15 long tag 327 328 TEM 16 long tag TEM 16 long tag 328 329TEM 17 long tag TEM 17 long tag 329 330 TEM 19 long tag TEM 19 long tag330 331 TEM 21 long tag TEM 21 long tag 331 332 TEM 21 long tag TEM 21long tag 332 333 TEM 22 long tag TEM 22 long tag 333 334 TEM 22 long tagTEM 22 long tag 334 335 TEM 23 long tag TEM 23 long tag 335 336 TEM 24long tag TEM 24 long tag 336 337 TEM 25 long tag TEM 25 long tag 337 338TEM 25 long tag TEM 25 long tag 338 339 TEM 28 long tag TEM 28 long tag339 340 TEM 30 long tag TEM 30 long tag 340 341 TEM 31 long tag TEM 31long tag 341 342 TEM 32 long tag TEM 32 long tag 342 343 TEM 33 long tagTEM 33 long tag 343 344 TEM 33 long tag TEM 33 long tag 344 345 TEM 35long tag TEM 35 long tag 345 346 TEM 36 long tag TEM 36 long tag 346 347TEM 37 long tag TEM 37 long tag 347 348 TEM 38 long tag TEM 38 long tag348 349 TEM 38 long tag TEM 38 long tag 349 350 TEM 39 long tag TEM 39long tag 350 351 TEM 40 long tag TEM 40 long tag 351 352 TEM 41 long tagTEM 41 long tag 352 353 TEM 42 long tag TEM 42 long tag 353 354 TEM 43long tag TEM 43 long tag 354 355 TEM 44 long tag TEM 44 long tag 355 356TEM 45 long tag TEM 45 long tag 356 357 TEM 46 long tag TEM 46 long tag357 358 TEM 35 Protein

1. An isolated and purified nucleic acid molecule comprising thesequence as shown in SEQ ID NO: 173, said sequence encoding atransmembrane TEM1.
 2. An isolated and purified nucleic acid moleculecomprising the coding sequence for a transmembrane TEM1 as shown in SEQID NO:
 177. 3. An isolated and purified nucleic acid molecule comprisingthe coding sequence for an extracellular domain of TEM1 comprising aminoacid residues 1 to 685 as shown in SEQ ID NO:
 177. 4. The isolated andpurified nucleic acid molecule of claim 3 which consists of amino acidresidues 1 to 685 as shown in SEQ ID NO:
 177. 5. The isolated andpurified nucleic acid molecule of claim 3 which comprises nucleotides 6to 2060 as shown in SEQ ID NO:
 173. 6. The isolated and purified nucleicacid molecule of claim 3 which consists of nucleotides 6 to 2060 asshown in SEQ ID NO:
 173. 7. A cultured host cell comprising a vectorcomprising a cDNA which encodes an immunogenic agent, wherein the hostcell expresses said immunogenic agent, wherein the immunogenic agentcomprises the extracellular domain of TEM1 comprising amino acidresidues 1 to 685 as shown in SEQ ID NO:
 177. 8. The host cell of claim7 wherein the cDNA comprises the sequence shown in SEQ ID NO:
 173. 9.The host cell of claim 7 wherein the cDNA comprises the coding sequencefor a transmembrane TEM1 as shown in SEQ ID NO:
 177. 10. The host cellof claim 7 wherein the cDNA consists of a coding sequence for amino acidresidues 1 to 685 as shown in SEQ ID NO:
 177. 11. The host cell of claim7 wherein the cDNA comprises nucleotides 6 to 2060 as shown in SEQ IDNO:
 173. 12. The host cell of claim 7 wherein the cDNA consists ofnucleotides 6 to 2060 as shown in SEQ ID NO:
 173. 13. A method of makinga TEM1 protein preparation, comprising: culturing a host cell comprisinga vector comprising a cDNA which encodes an immunogenic agent, whereinthe host cell expresses said immunogenic agent, wherein the immunogenicagent comprises the extracellular domain of TEM1 comprising amino acidresidues 1 to 685 as shown in SEQ ID NO: 177; isolating proteinexpressed by the host cell from other cellular constituents.
 14. Themethod of claim 13 wherein the cDNA comprises the sequence shown in SEQID NO:
 173. 15. The method of claim 13 wherein the cDNA comprises thecoding sequence for a transmembrane TEM1 as shown in SEQ ID NO:
 177. 16.The method of claim 13 wherein the cDNA comprises the coding sequencefor an extracellular domain of TEM1 comprising amino acid residues 1 to685 as shown in SEQ ID NO:
 177. 17. The method of claim 13 wherein thecDNA comprises nucleotides 6 to 2060 as shown in SEQ ID NO:
 173. 18. Themethod of claim 13 wherein the cDNA consists of nucleotides 6 to 2060 asshown in SEQ ID NO:
 173. 19. A cultured host cell comprising a vectorcomprising a cDNA which encodes an immunogenic agent, wherein the hostcell expresses said immunogenic agent, wherein the immunogenic agent isselected from the group consisting of TEM 1 protein as shown in SEQ IDNO: 177 and an immunogenic fragment of said TEM1 protein.
 20. A methodof making a TEM 1 protein preparation, comprising: culturing a host cellcomprising a vector comprising a cDNA encoding an immunogenic agentwhereby said immunogenic agent is expressed in the host cell, whereinthe immunogenic agent is selected from the group consisting of TEM1protein as shown in SEQ ID NO: 177 and an immunogenic fragment of saidTEM1 protein; isolating protein expressed by the host cell from othercellular constituents.